Modulators

ABSTRACT

Antibodies that modulate insulin receptor signaling are provided.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a divisional of U.S. patent application Ser.No. 12/890,598, filed Sep. 24, 2010, now U.S. Pat. No. 8,926,976, issuedJan. 6, 2015, which claims the priority benefit of U.S. ProvisionalPatent Application No. 61/246,067, filed Sep. 25, 2009, U.S. ProvisionalPatent Application No. 61/306,321, filed Feb. 19, 2010, and U.S.Provisional Patent Application No. 61/358,749, filed Jun. 25, 2010, eachof which is incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present disclosure relates to novel modulators and/or agonists ofthe insulin-insulin receptor signaling complex and to methods ofscreening for such modulators and/or agonists. Such modulators and/oragonists may, for example, be used to treat a mammalian subjectsuffering from Type 2 diabetes, obesity, hyperglycemia,hyperinsulinemia, insulin overdose, chronic kidney disease, Type 1diabetes, insulin resistance and disease states and conditionscharacterized by insulin resistance or to prevent occurrence of the samein an at risk subject.

BACKGROUND OF THE INVENTION

The present disclosure relates to novel modulators and/or agonists ofthe insulin-insulin receptor signaling complex, to methods of screeningfor such modulators and/or agonists and to the use of such modulatorsand/or agonists in the treatment or prevention of disease states andconditions characterized by abnormal production and/or utilization ofinsulin.

The peptide hormone insulin is a major regulator of glucose homeostasisand cell growth. The first step in insulin action is the binding of thehormone to the insulin receptor (INSR), an integral membraneglycoprotein, also designated as CD220 or HHF5. The INSR belongs to thetyrosine kinase growth factor receptor superfamily and is composed oftwo extracellular a subunits that bind insulin, and two transmembrane βsubunits with intrinsic tyrosine kinase activity. The amino acidsequence of the INSR is described in U.S. Pat. No. 4,761,371 and as NCBIReference Sequence NP_000199.2. The INSR is expressed in two isoforms,INSR-A and INSR-B. The three-dimensional structure of the intacthomodimeric ectodomain fragment of human INSR has been elucidated usingX-ray crystallography (WO07/147213). INSR isoforms also formheterodimers, INSR-A/INSR-B, and hybrid INSR/IGF-1R receptors, whoserole in physiology and disease is not yet fully understood (Belfiore etal, Endocrine Rev., 30(6):586-623, 2009).

When insulin binds to the INSR, the receptor is activated by tyrosineautophosphorylation and the INSR tyrosine kinase phosphorylates variouseffector molecules, including the insulin receptor substrate-1 (IRS-1),leading to hormone action (Ullrich et al, Nature 313: 756-761, 1985;Goldfine et al, Endocrine Reviews 8: 235-255, 1987; White and Kahn,Journal Biol. Chem. 269: 1-4, 1994). IRS-1 binding and phosphorylationeventually leads to an increase in the high affinity glucose transporter(Glut4) molecules on the outer membrane of insulin-responsive tissues,including muscle cells and adipose tissue, and therefore to an increasein the uptake of glucose from blood into these tissues. Glut4 istransported from cellular vesicles to the cell surface, where it thencan mediate the transport of glucose into the cell. A decrease in INSRsignaling, leads to a reduction in the uptake of glucose by cells,hyperglycemia (an increase in circulating glucose), and all the sequelaewhich result.

Reduction in glucose uptake can result in insulin resistance, whichdescribes a condition in which physiological amounts of insulin areinadequate to produce a normal insulin response from cells or tissues.Severe insulin resistance is associated with diabetes, while less severeinsulin resistance is also associated with a number of disease statesand conditions present in approximately 30-40% of non-diabeticindividuals (reviewed in Woods et al, End, Metab & Immune Disorders—DrugTargets 9: 187-198, 2009).

Current treatments for diabetes and insulin resistance are directedtoward improving insulin secretion, reducing glucose production, andenhancing insulin action.

Currently, there are various pharmacological approaches for thetreatment of Type 2 diabetes (Scheen et al, Diabetes Care,22(9):1568-1577, 1999; Zangeneh et al, Mayo Clin. Proc. 78: 471-479,2003; Mohler et al, Med Res Rev 29(1): 125-195, 2009). They act viadifferent modes of action: 1) sulfonylureas (e.g., glimepiride,glisentide, sulfonylurea, AY31637) essentially stimulate insulinsecretion; 2) biguanides (e.g., metformin) act by promoting glucoseutilization, reducing hepatic glucose production and diminishingintestinal glucose output; 3) alpha-glucosidase inhibitors (e.g.,acarbose, miglitol) slow down carbohydrate digestion and consequentlyabsorption from the gut and reduce postprandial hyperglycemia; 4)thiazol-idinediones (e.g., troglitazone, pioglitazone, rosiglitazone,glipizide, balaglitazone, rivoglitazone, netoglitazone, troglitazone,englitazone, AD 5075, T 174, YM 268, R 102380, NC 2100, NIP 223, NIP221, MK 0767, ciglitazone, adaglitazone, CLX 0921, darglitazone, CP92768, BM 152054) enhance insulin action, thus promoting glucoseutilization in peripheral tissues; 5) glucagon-like-peptides andagonists (e.g. exendin) or stabilizers thereof (e.g. DPP4 inhibitors,such as sitagliptin) potentiate glucose-stimulated insulin secretion;and 6) insulin or analogues thereof (e.g. LANTUS®) stimulate tissueglucose utilization and inhibits hepatic glucose output. The abovementioned pharmacological approaches may be utilized individually or incombination therapy. However, each approach has its limitations andadverse effects. Over time, a large percentage of Type 2 diabeticsubjects lose their response to these agents. 63% of Type 2 diabetespatients fail to reach global HbA_(1c) levels of <7% as advised by theAmerican Diabetes Association, and are thus at high risk of developingcomplications. Moreover, almost invariably patients progress through thestages of declining pancreatic function. Insulin treatment is typicallyinstituted after diet, exercise, and oral medications have failed toadequately control blood glucose. The drawbacks of insulin treatment arethe need for drug injection, the potential for hypoglycemia, and weightgain. Consequently there is still an urgent need for novel anti-diabeticagents.

Antibodies binding to human INSR have been reported in Soos et al,Biochem. J. 235: 199-208, 1986; Taylor et al, Biochem. J. 242: 123-129,1987; Prigent et al, J. Biol. Chem. 265(17):9970-9977, 1990; Brindle etal, Biochem. J. 268: 615-620, 1990; Steele-Perkins and Roth, J. Biol.Chem. 265(16): 9458-9463, 1990; McKern et al, Nature 443(14): 218-221;Boado et al, Biotech and Bio Eng. 96(2): 381-391; WO04/050016; Roth etal, Proc. Natl. Acad. Sci. USA 79: 7312-7316, 1982; Morgan et al, Proc.Natl. Acad. Sci. USA 83: 328-332, 1986; Lebrun et al, J. Bl. Chem.268(15): 11272-11277, 1993; Forsayeth et al, Proc. Natl. Acad. Sci. USA84: 3448-3451, 1987; Forsayeth et al, J. Biol. Chem. 262(9): 4134-4140,Goodman et al, J. Receptor Res. 14(6-8), 381-398, 1994; Ganderton et al,Biochem J. 288: 195-205, 1992; Spasov et al, Bull. of Exp. Biol. andMed. 144(1): 46-48, 2007; EP 2 036 574 A1.

SUMMARY OF THE INVENTION

The present disclosure is directed to polypeptide binding agents, e.g.,antibodies or fragments thereof, that modulate and/or agonize theinsulin-INSR signaling complex by binding to extracellular regions ofthe INSR uncomplexed to inslin, to the INSR complexed with insulin, orto both. INSR is a membrane-bound cell surface receptor.

In one aspect, the invention provides an antibody that binds to insulinreceptor and/or a complex comprising insulin and insulin receptor withan equilibrium dissociation constant K_(D) of 10⁻⁵M or less that iscapable of strengthening binding affinity or binding rate parameterbetween insulin and insulin receptor (INSR) by about 5-fold to 200-fold.In one embodiment, the antibody is capable of strengthening the bindingaffinity or binding rate parameter between insulin and insulin receptorby about 1.5-fold to about 100-fold, or about 2-fold to 25-fold. It isfurther contemplated that the modulation is about 2-fold to about50-fold, or about 1.5-fold to about 25-fold, or about 1.5-fold to about50-fold, e.g., at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold,6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold,14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold or 20-fold, or upto 100-fold, or up to 90-fold, or up to 80-fold, or up to 70-fold, or upto 60-fold, or up to 50-fold, or up to 40-fold, or up to 30-fold, or upto 20-fold, or up to 10-fold. In a further embodiment, the antibodystrengthens binding affinity or binding rate parameter by 1.5, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100fold or more, or any range between any of these values. In someembodiments, the binding affinity is any one of K_(A), K_(D), the ratioof on rate divided by off rate, or the ratio of off rate divided by onrate. In specific exemplary embodiments, the antibody increases K_(A) bythe desired fold, or decreases K_(D) by the desired fold, or increasesthe ratio of on rate to off rate by the desired fold, or decreases theratio of off rate to on rate by the desired fold. In some embodiments,the binding rate parameter is the on rate or off rate. In specificexemplary embodiments, the antibody increases the on rate or decreasesthe off rate. Alternatively, in some embodiments where the bindingaffinity does not change detectably or significantly, increasing the onrate and increasing the off rate may shift the signaling pathway awayfrom mitogenic signaling towards metabolic signaling (glucose uptake).

In one embodiment, an antibody that strengthens the binding affinitybetween insulin and INSR is a positive modulator.

In another aspect, the antibody is an agonist.

In a related aspect, an antibody that activates the INSR withoutdependence on insulin is an allosteric agonist. In certain embodiments,the invention provides an allosteric agonist antibody that binds toinsulin receptor with an affinity of at least 10⁻⁵, 10⁻⁶, 10⁻⁷, 10⁻⁸,10⁻⁹ M and (a) exhibits maximal agonist activity that is 20%-80% that ofinsulin's maximal agonist activity when measured in a pAKT assay, (b)when present does not alter the EC50 of insulin for INSR by more than2-fold, and (c) when present does not alter the K_(D) of insulin forINSR by more than 2-fold.

In a related embodiment, the allosteric agonist exhibits a maximalagonist response that is 80% or less of the maximal agonist response ofinsulin, for example 15%-80%, 20-60%, 20%-40% or 15%-30%. In certainembodiments, the antibodies constitutively activate INSR with a maximalagonist response that is at least about 15%, 20%, 25%, 30%, 35%, 40%;and up to 45%, 50%, 55%, 60%, 65%, 70%, 75% or 80% of the maximalagonist response of insulin. It is understood that any combination ofany of these range endpoints is contemplated without having to reciteeach possible combination.

In another aspect, the invention provides an antibody that binds toinsulin receptor and/or a complex comprising insulin and insulinreceptor with an equilibrium dissociation constant K_(D) of 10⁻⁵M orless that is capable of weakening the binding affinity or binding rateparameter between insulin and insulin receptor by about 1.5-fold to100-fold. In one embodiment, the antibody is capable of weakening thebinding affinity or binding rate parameter between insulin and insulinreceptor by about 2-fold to 25-fold, or 1.5-fold to 25 fold, or 2-foldto 50-fold. It is further contemplated that the modulation is about2-fold to about 50-fold, or about 1.5-fold to about 25-fold, or about1.5-fold to about 50-fold, e.g. at least 1.5-fold, 2-fold, 3-fold,4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold,12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-foldor 20-fold, or up to 100-fold, or up to 90-fold, or up to 80-fold, or upto 70-fold, or up to 60-fold, or up to 50-fold, or up to 40-fold, or upto 30-fold, or up to 20-fold, or up to 10-fold. In a further embodiment,the antibody weakens binding affinity or binding rate parameter by 1.5,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95or 100 fold or more, or any range between any of these values. In someembodiment, the binding affinity is any one of K_(A), K_(D), the ratioof on rate divided by off rate, or the ratio of off rate divided by onrate. In specific exemplary embodiments, the antibody decreases K_(A) bythe desired fold, or increases K_(D) by the desired fold, or decreasesthe ratio of on rate to off rate by the desired fold, or increases theratio of off rate to on rate by the desired fold. In some embodiments,the binding rate parameter is the on rate or off rate. In specificexemplary embodiments, the antibody decreases the on rate or increasesthe off rate.

In one embodiment, an antibody that weakens the binding affinity betweeninsulin and INSR is a negative modulator. In some specific embodiments,an antibody that weakens the binding affinity between insulin and INSRis an antagonist.

In still another embodiment, an antibody that strengthens or weakensbinding affinity or binding rate parameter between insulin and insulinreceptor comprises at least one heavy chain CDR (HCDR1, HCDR2 and HCDR3)set out in SEQ ID NOS: 151-303. In a related embodiment, the antibodycomprises a mature heavy chain variable region of SEQ ID NO: 151-303. Itis contemplated that any of the above antibodies further comprises asuitable human or human consensus or human-derived constant region, e.g.IgG1, IgG2, IgG3, or IgG4 or a hybrid thereof.

In a further embodiment, the antibody that strengthens or weakensbinding affinity or binding rate parameter between insulin and insulinreceptor comprises at least one light chain CDR (LCDR1, LCDR2, or LCDR3)set out in SEQ ID NOS: 1-150. In still another embodiment, the antibodycomprises a mature light chain variable region of SEQ ID NO: 1-150. Itis contemplated that any of the above antibodies further comprises ahuman kappa or lambda light chain constant region.

In one embodiment, the antibody binds insulin receptor. In a relatedembodiment, the antibody binds the α subunit of INSR. In a furtherembodiment, the antibody binds the β subunit of INSR. In yet anotherembodiment, the antibody binds the α and β subunit of the receptor. In arelated embodiment, the antibody binds an insulin/insulin receptorcomplex. In another embodiment, the antibody that binds the insulin/INSRcomplex does not detectably bind insulin receptor alone, e.g., in theabsence of insulin, or insulin alone.

In another aspect, the invention provides an antibody that specificallybinds insulin receptor and/or a complex comprising insulin and insulinreceptor with an equilibrium dissociation constant K_(D) of 10⁻⁵M orless, comprising at least one heavy chain CDR (HCDR1, HCDR2 and HCDR3)of SEQ ID NOS: 151-303. In a related embodiment, the antibody comprisesa heavy chain variable region of SEQ ID NO: 151-303. It is contemplatedthat any of the above antibodies further comprises a suitable human orhuman consensus or human-derived constant region, e.g. IgG1, IgG2, IgG3,or IgG4 or a hybrid thereof.

In a further embodiment, the antibody that specifically binds insulinreceptor and/or a complex comprising insulin and insulin receptor withan equilibrium dissociation constant K_(D) of 10⁻⁵M or less, comprisesat least one light chain CDR (LCDR1, LCDR2, or LCDR3) set out in SEQ IDNOS: 1-150. In still another embodiment, the antibody comprises a lightchain variable region of SEQ ID NO: 1-150. It is contemplated that anyof the above antibodies further comprises a human kappa or lambda lightchain constant region.

It is further contemplated that any of the antibodies described abovecomprises one, two, three, four, five or six CDRs. In one embodiment,the antibody comprises one, two or three heavy chain CDRs set out in SEQID NO: 151-303. In another embodiment, the antibody comprises one, twoor three light chain CDRs set out in SEQ ID NO: 1-150.

In one embodiment, the antibody binds insulin receptor. In a relatedembodiment, the antibody binds an insulin/INSR complex. In anotherembodiment, the antibody that binds the insulin/INSR complex does notdetectably bind insulin receptor alone or insulin alone.

It is further contemplated that an antibody that strengthens bindingaffinity or binding rate parameter of insulin and insulin receptoractivates insulin receptor by at least 10% of the maximal signal ofinsulin, optionally in a phosphorylated AKT assay. In a relatedembodiment, the INSR is activated by at least 11%, 12%, 13%, 14%, 15%,16%, 17%, 18%, 19%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, or 80% of themaximal signal of insulin. In another embodiment, the antibody thatstrengthens binding affinity or binding rate parameter of insulin/INSRactivates less than 10% of the maximal signal of insulin, optionally ina phosphorylated AKT assay. In a related embodiment, the INSR isactivated by less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of themaximal signal of insulin. In some embodiments, the INSR is notdetectably activated by the antibody.

It is further contemplated that the antibody reduces fasting bloodglucose levels, in a subject having elevated blood glucose,hyperglycemia or disorder associated with insulin resistance, toward thenormal range of glucose levels. In one embodiment, the positivemodulating antibody or agonist reduces fasting blood glucose levels inthe subject by approximately 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% ormore.

In one embodiment, antibody refers to an antibody or fragment thereof,or a polypeptide comprising an antigen binding domain of an antibody.Exemplary antibodies or antibody fragments include polyclonalantibodies, monoclonal antibodies, chimeric antibodies, humanizedantibodies, human antibodies, multispecific antibodies, Fab, Fab′,F(ab′)2, Fv, domain antibody (dAb), complementarity determining region(CDR) fragments, CDR-grafted antibodies, single-chain antibodies (scFv),single chain antibody fragments, chimeric antibodies, diabodies,triabodies, tetrabodies, minibody, linear antibody; chelatingrecombinant antibody, a tribody or bibody, an intrabody, a nanobody, asmall modular immunopharmaceutical (SMIP), a antigen-binding-domainimmunoglobulin fusion protein, a camelized antibody, a VHH containingantibody, or a variant or a derivative thereof, and polypeptides thatcontain at least a portion of an immunoglobulin that is sufficient toconfer specific antigen binding to the polypeptide, such as one, two,three, four, five or six CDR sequences. In one embodiment, the antibodyis a monoclonal antibody. In a related embodiment, the antibody is ahuman antibody.

In some specific embodiments, the invention excludes rodent antibodies,i.e. antibodies produced by a hybridoma of rodent (e.g. murine, rat)cells. Such antibodies, whether produced by the hybridoma orrecombinantly, would have rodent framework amino acid sequence and beimmunogenic if administered to humans. In some specific embodiments, theinvention excludes the rodent antibodies disclosed in any one of thefollowing references, hereby incorporated by reference in theirentirety: Soos et al, Biochem. J. 235: 199-208, 1986; Taylor et al,Biochem. J. 242: 123-129, 1987; Prigent et al, J. Biol. Chem.265(17):9970-9977, 1990; Brindle et al, Biochem. J. 268: 615-620, 1990;Steele-Perkins and Roth, J. Biol. Chem. 265(16): 9458-9463, 1990; McKernet al, Nature 443(14): 218-221; Boado et al, Biotech and BioEng. 96(2):381-391; WO04/050016; Roth et al, Proc. Natl. Acad. Sci. USA 79:7312-7316, 1982; Morgan et al, Proc. Natl. Acad. Sci. USA 83: 328-332,1986; Lebrun et al, J. Bl. Chem. 268(15): 11272-11277, 1993; Forsayethet al, Proc. Natl. Acad. Sci. USA 84: 3448-3451, 1987; Forsayeth et al,J. Biol. Chem. 262(9): 4134-4140, Goodman et al, J. Receptor Res.14(6-8), 381-398, 1994; Ganderton et al, Biochem J. 288: 195-205, 1992;Spasov et al, Bull. of Exp. Biol. and Med. 144(1): 46-48, 2007; EP 2 036574 A1. However, the invention may include humanized versions of suchrodent antibodies, treatment methods using such humanized antibodies,and sterile pharmaceutical compositions comprising such humanizedantibodies. In some specific embodiments, the invention excludes thehumanized antibody 83-14 reported in Boado et al, Biotech and BioEng.96(2): 381-391 or WO04/050016.

In exemplary embodiments, the invention contemplates:

a monoclonal antibody that retains any one, two, three, four, five, orsix of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3 of any one of SEQ IDNOs: 151-303 and SEQ ID NOs: 1-150, respectively, optionally includingone or two mutations in such CDR(s), e.g., a conservative ornon-conservative substitution, and optionally paired as set forth inTable 3;

a monoclonal antibody that retains all of HCDR1, HCDR2, HCDR3, or theheavy chain variable region of any one of SEQ ID NOs: 151-303,optionally including one or two mutations in any of such CDR(s),optionally further comprising any suitable heavy chain constant region,e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, or IgE or hybrid thereof;

a monoclonal antibody that retains all of LCDR1, LCDR2, LCDR3, or thelight chain variable region of any one SEQ ID NOs: 1-150, optionallyincluding one or two mutations in any of such CDR(s), optionally furthercomprising to any suitable light chain constant region, e.g. a kappa orlambda light chain constant region;

a purified preparation of a monoclonal antibody, comprising the lightchain variable region and heavy chain variable regions as set forth inSEQ ID NOs: 1-303 and paired as set forth in Table 3;

a monoclonal antibody that binds to the same linear or three-dimensionalepitope of INSR as an antibody comprising a variable region set out inSEQ ID NO: 1-303 a, e.g., as determined through X-ray crystallography orother biophysical or biochemical techniques such as deuterium exchangemass spectrometry, alanine scanning and peptide fragment ELISA;

a monoclonal antibody that competes with an antibody comprising avariable region set out in SEQ ID NO: 1-303, optionally paired as inTable 3, for binding to human INSR by more than about 75%, more thanabout 80%, or more than about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94% or 95%.

In some embodiments, the antibody comprises all three light chain CDRs,all three heavy chain CDRs, or all six CDRs of an antibody comprising avariable region set out in SEQ ID NO: 1-303. In some exemplaryembodiments, two light chain CDRs from an antibody may be combined witha third light chain CDR from a different antibody. Alternatively, aLCDR1 from one antibody can be combined with a LCDR2 from a differentantibody and a LCDR3 from yet another antibody, particularly where theCDRs are highly homologous. Similarly, two heavy chain CDRs from anantibody may be combined with a third heavy chain CDR from a differentantibody; or a HCDR1 from one antibody can be combined with a HCDR2 froma different antibody and a HCDR3 from yet another antibody, particularlywhere the CDRs are highly homologous.

Consensus CDRs may also be used. Any one of the consensus CDRs derivedherein may be combined with two other CDRs from the same chain (e.g.,heavy or light) of any of the antibodies described herein, e.g. to forma suitable heavy or light chain variable region.

In another aspect, the invention provides variants or derivatives of theantibodies described herein. For example, in one embodiment the antibodyis labeled with a detectable moiety as described herein. In a furtherembodiment, the antibody is conjugated to a hydrophobic moiety describedherein.

Variants of the antibodies include antibodies having a mutation oralteration in an amino acid sequence provided herein, including an aminoacid insertion, deletion or substitution, e.g., a conservative ornon-conservative substitution.

In some embodiments, an antibody is provided that comprises apolypeptide having an amino acid sequence at least about 65%, 70%, 75%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more identical to the heavy chainvariable region set out in SEQ ID NO: 151-303 and/or an amino acidsequence an amino acid sequence at least about 65%, 70%, 75%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more identical to the light chain variable regionset out in SEQ ID NO: 1-150, the antibody further comprising at leastone, two, three, four, five or all of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2or CDRL3. In some embodiments, the amino acid sequence with percentageidentity to the light chain variable region may comprise one, two orthree of the light chain CDRs. In other embodiments, the amino acidsequence with percentage identity to the heavy chain variable region maycomprise one, two, or three of the heavy chain CDRs.

It is contemplated that the antibodies of the invention may have one, ortwo or more amino acid substitutions in the CDR regions of the antibody,e.g. conservative substitutions.

In a related embodiment, the residues of the framework are altered. Theheavy chain framework regions which can be altered lie within regionsdesignated H-FR1, H-FR2, H-FR3 and H-FR4, which surround the heavy chainCDR residues, and the residues of the light chain framework regionswhich can be altered lie within the regions designated L-FR1, L-FR2,L-FR3 and L-FR4, which surround the light chain CDR residues. An aminoacid within the framework region may be replaced, for example, with anysuitable amino acid identified in a human framework or human consensusframework.

It is further contemplated that the invention provides a purifiedpolypeptide comprising any one of the amino acid sequences of SEQ ID NO:1-150 fused to any one of the amino acid sequences of SEQ ID NO:151-303, optionally paired as the heavy/light chain variable regions setforth in Table 3, or fragments thereof that include at least a portionof SEQ ID NO: 1-150 and SEQ ID NO: 151-303, optionally paired as setforth in Table 3, wherein the polypeptide binds insulin receptor,insulin or the insulin/insulin receptor complex.

It is contemplated that antibodies of the invention, includingpolypeptides comprising all or a portion of an antigen binding fragmentin any one of SEQ ID NOs: 1-303, retain binding affinity, e.g. asmeasured by K_(D), to insulin receptor, insulin or a complex ofinsulin/INSR of 10⁻⁵, 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹ M or less (wherein a lowervalue indicates a higher binding affinity), optionally as measured bysurface plasmon resonance.

In some of the preceding embodiments, the invention contemplates anantibody that binds to insulin receptor and/or a complex comprisinginsulin and insulin receptor, with an equilibrium dissociation constantK_(D) of 10⁻⁵M or less, that is capable of strengthening the bindingaffinity between insulin and insulin receptor by about 5-fold to500-fold. In one embodiment, the antibody is characterized by thefollowing equilibrium dissociation constant K_(D) binding properties:(i) said antibody binds with an equilibrium dissociation constant K_(D)of about 10⁻⁵M, 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹ 10⁻¹⁰, 10⁻¹¹ M or less, to acomplex comprising insulin (C1) or and insulin receptor (C2); and (ii)any of K_([C1C2]A), K_([AC2]C1), or K_([AC1]C2) is at least about 5-foldlower than any of K_(AC2) or K_(AC1). In a related embodiment, any ofK_([C1C2]A), K_([AC2]C1), or K_([AC1]C2) is about 5-fold to 200-foldlower than any of K_(AC2) or K_(AC1).

In some embodiments, the antibody binds an insulin/insulin receptorcomplex. In further embodiments, the antibody binds insulin receptoralone, in an uncomplexed form. In a related embodiment, the antibodydoes not detectably bind insulin receptor alone, e.g., in the absence ofinsulin. In certain embodiments, the antibody is capable ofstrengthening the binding affinity between insulin and insulin receptorby at least about 5-fold, optionally to about 200-fold, optionally toabout 100-fold. It is further provided that in some embodiments, thebinding affinity is any one of K_(A), K_(D), the ratio of on rate to offrate, or the ratio of off rate to on rate.

In some embodiments, for any of the antibodies described herein, thedifference in binding affinity or binding rate parameter ranges fromabout 1.5-fold to about 1000-fold, or about 1.5-fold to about 500-fold,about 1.5-fold to about 100-fold, or about 2-fold to 25-fold, or about2-fold to about 50-fold, or about 1.5-fold to about 25-fold, or about1.5-fold to about 50-fold, about 5-fold to about 500-fold, or about5-fold to about 200-fold, e.g. at least about 1.5-fold, 2-fold, 3-fold,4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold,12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-foldor 20-fold, or up to 500-fold, or up to 200-fold, or up to 150-fold, orup to 100-fold, or up to 90-fold, or up to 80-fold, or up to 70-fold, orup to 60-fold, or up to 50-fold, or up to 40-fold, up to 30-fold, up to20-fold, or up to 10-fold, or up to 5-fold or up to 3-fold.

In some embodiments, the invention provides an agonist antibody thatbinds to insulin receptor with an affinity, e.g. K_(D), of 10⁻⁵, 10⁻⁶,10⁻⁷, 10⁻⁸, 10⁻⁹ 10⁻¹⁰, 10⁻¹¹ M or less, optionally that exhibitsmaximal agonist activity that is 20%-100% that of insulin's maximalagonist activity when measured in pAKT assay. In a related aspect, theinvention contemplates an allosteric agonist antibody that binds toinsulin receptor with an affinity, e.g., K_(D) of 10⁻⁵, 10⁻⁶, 10⁻⁷,10⁻⁸, 10⁻⁹ 10⁻¹⁰, 10⁻¹¹ M or less and (a) exhibits maximal agonistactivity that is 20%-80% that of insulin's maximal agonist activity whenmeasured in pAKT assay, (b) when present does not alter the EC50 ofinsulin for INSR by more than 2-fold or 3-fold, and (c) when presentdoes not alter the K_(D) of insulin for INSR by more than 2-fold or3-fold. It is further provided that in some embodiments, the bindingaffinity is any one of K_(A), K_(D), the ratio of on rate to off rate,or the ratio of off rate to on rate.

It is contemplated, in certain embodiments, that any of the aboveantibodies may also exhibit weak agonist activity, e.g., activatesinsulin receptor by at least 10% of the maximal signal of insulin,optionally in a phosphorylated AKT assay. In a further embodiment, theantibody activates insulin receptor by less than 10% of the maximalsignal of insulin, optionally in a phosphorylated AKT assay.

In some embodiments, the antibody comprises a heavy chain variableregion selected from the group consisting of the mature heavy chainvariable region sequences set forth in SEQ ID NOs: 281, 278, 277, 209,275, 223, 284, 276, and 236 and a light chain variable region selectedfrom the group consisting of the mature light chain variable sequencesset forth in SEQ ID NOs: 141, 138, 137, 35, 135, 57, 144, 136, and 98,optionally paired as set forth in Table 3.

In another embodiment, the antibody comprises (a) the heavy chainvariable region of any of Ab006, Ab030, Ab004, Ab013, Ab009, Ab007,Ab011, Ab001, Ab012, Ab010, Ab003, Ab008, Ab002, Ab005, Ab076, Ab077,Ab079, Ab080, Ab083, Ab059, Ab078, Ab085 or set out in SEQ ID NO: 291,196, 239, 267 and 271 and the light chain variable region of any ofAb006, Ab030, Ab004, Ab013, Ab009, Ab007, Ab011, Ab001, Ab012, Ab010,Ab003, Ab008, Ab002, Ab005, Ab076, Ab077, Ab079, Ab080, Ab083, Ab059,Ab078, Ab085 or set out in SEQ ID NO: 76, 80, 101, 128, and 132,optionally paired as set forth in Table 3 and preferably the matureportions thereof, or (b) one, two or three heavy chain CDRs of any ofAb006, Ab030, Ab004, Ab013, Ab009, Ab007, Ab011, Ab001, Ab012, Ab010,Ab003, Ab008, Ab002, Ab005, Ab076, Ab077, Ab079, Ab080, Ab083, Ab059,Ab078, Ab085 or set out in SEQ ID NO: 291, 196, 239, 267 and 271 and/orone, two or three light chain CDRs of any of Ab006, Ab030, Ab004, Ab013,Ab009, Ab007, Ab011, Ab001, Ab012, Ab010, Ab003, Ab008, Ab002, Ab005,Ab076, Ab077, Ab079, Ab080, Ab083, Ab059, Ab078, Ab085 or set out in SEQID NO: 76, 80, 101, 128, and 132, optionally including one or twomutations in any one, two or three of such heavy or light chain CDRs,e.g., a conservative or non-conservative substitution, optionally pairedas set forth in Table 3; or (c) all six CDRs of any of Ab006, Ab030,Ab004, Ab013, Ab009, Ab007, Ab011, Ab001, Ab012, Ab010, Ab003, Ab008,Ab002, Ab005, Ab076, Ab077, Ab079, Ab080, Ab083, Ab059, Ab078, Ab085 orantibodies having the variable regions set out in SEQ ID NO: 76, 80,101, 128, 132, 291, 196, 239, 267, and 271, optionally paired as setforth in Table 3.

In other embodiments, the invention provides an antibody that competeswith any of the antibodies described herein, e.g. by at least 70%, 75%,or 80%. In certain embodiments, the antibody exhibits greater than orequal to 70% competition, e.g. at least 75% or at least 80% competition,with any one, two, three or all antibodies selected from the groupconsisting of Ab079, Ab076, Ab083, Ab080, Ab062, Ab020, Ab019, Ab088,and Ab089, and optionally exhibits greater than or equal to 30%competition with any one, two, three or all antibodies selected from thegroup consisting of Ab086, Ab064, Ab001, and Ab018. Optionally, theantibody does not compete with one or more of Ab062 and Ab086, andoptionally may bind both human and murine insulin receptor or complex.

In a related embodiment, the antibody exhibits greater than or equal to70% competition, e.g., at least 75% or at least 80% competition, withany one, two, three or all antibodies selected from the group consistingof Ab040, Ab062, Ab030, Ab001, and Ab018, and optionally exhibitsgreater than or equal to 30% competition with any one, two, three or allantibodies selected from the group consisting of Ab037, Ab078, Ab083,Ab080, and Ab085. In a related embodiment, the antibody does not competewith any one, two, three or more of antibodies selected from the groupconsisting of Ab053, Ab064, 83-7, Ab019, Ab088, and Ab089. Optionally,the antibody binds both human and murine insulin receptor or complex. Inanother embodiment, the antibody exhibits greater than or equal to 70%competition, e.g. at least 75% or at least 80% competition, with anyone, two, three or all antibodies selected from the group consisting ofAb064, Ab062, Ab085, and Ab078. Optionally, the antibody exhibits nocompetition with any one, two, three or more of antibodies selected fromthe group consisting of Ab077, Ab001, Ab018, Ab030, Ab037, Ab079, Ab076,Ab083, Ab019, Ab088, Ab089, and Ab040. Optionally, the antibody bindsboth human and murine insulin receptor or complex.

In a further aspect, the invention provides an antibody that binds toinsulin receptor and/or a complex comprising insulin and insulinreceptor with an equilibrium dissociation constant K_(D) of 10⁻⁵M orless that is capable of weakening the binding affinity between insulinand insulin receptor by at least about 3-fold, optionally up to1000-fold. In certain embodiments, the antibody weakens the affinitybetween said insulin and insulin receptor by about 3-fold to 500-fold.In some embodiments, the binding affinity is any one of K_(A), K_(D),the ratio of on rate to off rate, or the ratio of off rate to on rate.

In some embodiments, for any of the antibodies described herein, thedifference in binding affinity or binding rate parameter ranges fromabout 1.5-fold to about 1000-fold, or about 1.5-fold to about 500-fold,about 1.5-fold to about 100-fold, or about 2-fold to 25-fold, or about2-fold to about 50-fold, or about 1.5-fold to about 25-fold, or about1.5-fold to about 50-fold, about 5-fold to about 500-fold, or about5-fold to about 200-fold, e.g. at least about 1.5-fold, 2-fold, 3-fold,4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold,12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-foldor 20-fold, or up to 500-fold, or up to 200-fold, or up to 150-fold, orup to 100-fold, or up to 90-fold, or up to 80-fold, or up to 70-fold, orup to 60-fold, or up to 50-fold, or up to 40-fold, up to 30-fold, up to20-fold, or up to 10-fold, or up to 5-fold or up to 3-fold.

In a related embodiment, the antibody increases the EC50 of insulinsignaling activity by about 2-fold to 1000-fold, optionally in a pAKTassay. In certain embodiments, the antibody increases the EC50 by atleast about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100,150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800,850, 900, 950 or 1000-fold.

In certain embodiments, the antibody comprises a heavy chain variableregion selected from the group consisting of the mature heavy chainvariable region sequences set forth in SEQ ID NOs: 241, 279, 258, 155,and 228 and a light chain variable region selected from the groupconsisting of the mature light chain variable region sequences set forthin SEQ ID NOs: 103, 139, 119, 8, and 89, optionally paired as set forthin Table 3.

In a further embodiment, the antibody comprises (a) the heavy chainvariable region of any of Ab087, Ab019, Ab088, Ab089, Ab020, Ab050,Ab052, Ab055, Ab057, Ab061, Ab063, Ab065, Ab070, Ab072, Ab074, Ab081 andthe light chain variable region of any of Ab087, Ab019, Ab088, Ab089,Ab020, Ab050, Ab052, Ab055, Ab057, Ab061, Ab063, Ab065, Ab070, Ab072,Ab074, Ab081, preferably the mature portions thereof, or (b) one, two orthree heavy chain CDRs of any of Ab087, Ab019, Ab088, Ab089, Ab020,Ab050, Ab052, Ab055, Ab057, Ab061, Ab063, Ab065, Ab070, Ab072, Ab074,Ab081 and/or one, two or three light chain CDRs of any of Ab087, Ab019,Ab088, Ab089, Ab020, Ab050, Ab052, Ab055, Ab057, Ab061, Ab063, Ab065,Ab070, Ab072, Ab074, Ab081, optionally including one or two mutations inany one, two or three of such heavy or light chain CDRs, e.g., aconservative or non-conservative substitution; or (c) all six CDRs ofany of Ab087, Ab019, Ab088, Ab089, Ab020, Ab050, Ab052, Ab055, Ab057,Ab061, Ab063, Ab065, Ab070, Ab072, Ab074, Ab081.

In a further embodiment, the invention provides an antibody thatcompetes with the above antibodies, wherein the antibody exhibitsgreater than or equal to 70% competition, e.g., at least 75% or at least80% competition, with any one, two, three or all antibodies selectedfrom the group consisting of Ab079, Ab076, Ab083, Ab080, Ab062, andAb020, Ab019, Ab088, Ab089. Optionally, the antibody does not exhibitcompetition with any one, two, three or more of the antibodies selectedfrom the group consisting of Ab062, Ab086, Ab001, Ab018, Ab030, Ab037,Ab064; and optionally, the antibody is human reactive only, and does notbind murine insulin receptor or complex.

In one embodiment, the invention provides an antibody that is anagonist, wherein the antibody comprises a heavy chain variable regionselected from the group consisting of the mature heavy chain variableregion sequences set forth in SEQ ID NOs: 195, 220, 303, 197, 208, 243,245 and 251 and a light chain variable region selected from the groupconsisting of the mature light chain variable region sequences set forthin SEQ ID NOs: 77, 50, 90, 84, 34, 104, 106 and 112, optionally pairedas set forth in Table 3.

In a related embodiment, the antibody comprises (a) the heavy chainvariable region of any of Ab021, Ab029, Ab022, Ab017, Ab023, Ab024,Ab025, Ab026, Ab031, Ab035, Ab027, Ab036, Ab037, Ab028, Ab038, Ab039,Ab040, Ab041, Ab042, Ab032, Ab043, Ab044, Ab045, Ab046, Ab047, Ab018,Ab033, Ab048, Ab014, Ab015, Ab049, Ab034, Ab051, Ab053, Ab054, Ab056,Ab058, Ab062, Ab064, Ab066, Ab067, Ab068, Ab086, Ab069, Ab071, Ab073,Ab075, Ab082, Ab084 set out in SEQ ID NOs: 252, 253, 263, 265 and 269and the light chain variable region of any of Ab021, Ab029, Ab022,Ab017, Ab023, Ab024, Ab025, Ab026, Ab031, Ab035, Ab027, Ab036, Ab037,Ab028, Ab038, Ab039, Ab040, Ab041, Ab042, Ab032, Ab043, Ab044, Ab045,Ab046, Ab047, Ab018, Ab033, Ab048, Ab014, Ab015, Ab049, Ab034, Ab051,Ab053, Ab054, Ab056, Ab058, Ab062, Ab064, Ab066, Ab067, Ab068, Ab086,Ab069, Ab071, Ab073, Ab075, Ab082, Ab084 or set out in SEQ ID NOs: 7,113, 114, 124, 126 and 130, optionally paired as set forth in Table 3and preferably the mature portions thereof, or (b) one, two or threeheavy chain CDRs of any of Ab021, Ab029, Ab022, Ab017, Ab023, Ab024,Ab025, Ab026, Ab031, Ab035, Ab027, Ab036, Ab037, Ab028, Ab038, Ab039,Ab040, Ab041, Ab042, Ab032, Ab043, Ab044, Ab045, Ab046, Ab047, Ab018,Ab033, Ab048, Ab014, Ab015, Ab049, Ab034, Ab051, Ab053, Ab054, Ab056,Ab058, Ab062, Ab064, Ab066, Ab067, Ab068, Ab086, Ab069, Ab071, Ab073,Ab075, Ab082, Ab084 or set out in SEQ ID NOs: 252, 253, 263, 265 and 269and/or one, two or three light chain CDRs of any of Ab021, Ab029, Ab022,Ab017, Ab023, Ab024, Ab025, Ab026, Ab031, Ab035, Ab027, Ab036, Ab037,Ab028, Ab038, Ab039, Ab040, Ab041, Ab042, Ab032, Ab043, Ab044, Ab045,Ab046, Ab047, Ab018, Ab033, Ab048, Ab014, Ab015, Ab049, Ab034, Ab051,Ab053, Ab054, Ab056, Ab058, Ab062, Ab064, Ab066, Ab067, Ab068, Ab086,Ab069, Ab071, Ab073, Ab075, Ab082, Ab084 or set out in SEQ ID NOs: 7,113, 114, 124, 126 and 130, optionally including one or two mutations inany one, two or three of such heavy or light chain CDRs, e.g., aconservative or non-conservative substitution, optionally paired as setforth in Table 3; (c) all six CDRs of any of Ab021, Ab029, Ab022, Ab017,Ab023, Ab024, Ab025, Ab026, Ab031, Ab035, Ab027, Ab036, Ab037, Ab028,Ab038, Ab039, Ab040, Ab041, Ab042, Ab032, Ab043, Ab044, Ab045, Ab046,Ab047, Ab018, Ab033, Ab048, Ab014, Ab015, Ab049, Ab034, Ab051, Ab053,Ab054, Ab056, Ab058, Ab062, Ab064, Ab066, Ab067, Ab068, Ab086, Ab069,Ab071, Ab073, Ab075, Ab082, Ab084 or set out in SEQ ID NOs: 7, 113, 114,124, 126, 130, 252, 253, 263, 265 and 269, optionally paired as setforth in Table 3.

In a related embodiment, the invention provides an antibody thatcompetes with the above antibodies for binding to target, wherein theantibody exhibits greater than or equal to 70% competition, e.g., atleast 75% or at least 80% competition, with any one, two, three or allantibodies selected from the group consisting of Ab030, Ab037, Ab053,Ab001, Ab018, Ab064, Ab040 and optionally exhibits greater than or equalto 30% competition with any one, two, three or all antibodies selectedfrom the group consisting of Ab085 and Ab086. Optionally, the antibodyexhibits no competition with any one, two, three or more of theantibodies selected from the group consisting of Ab079, Ab076 and Ab088;and optionally binds to both human and murine insulin receptor orcomplex.

In a another aspect, the invention provides polynucleotides encodingantibodies and polypeptides of the invention, vectors comprising suchpolynucleotides, host cells comprising such polynucleotides or vectors,and methods of producing antibodies and polypeptides of the inventioncomprising growing such host cells in culture medium under suitableconditions and optionally isolating the encoded antibody or polypeptidefrom the host cells or culture medium, optionally followed by furtherpurification of the antibody or polypeptide, e.g., as described herein.

Antibodies having the properties described herein may be isolated usinga screening method to determine binding to the INSR and modulation ofthe insulin/INSR complex.

In one embodiment, the invention provides a positive modulating antibodythat strengthens the binding of a first component (C1) to a secondcomponent (C2) of a signaling complex, said antibody characterized bythe following equilibrium dissociation constant K_(D) bindingproperties: (i) said antibody binds with an equilibrium dissociationconstant K_(D) of about 10⁻⁵M or less, e.g., 10⁻⁶M or less, or 10⁻⁷M orless, or 10⁻⁸M or less, to any one of C1, C2, or a complex comprising C1and C2 (C1C2); and (ii) any of K_([C1C2]A), K_([AC2]C1), or K_([AC1]C2)is at least about 50% (1.5-fold) lower than any of K_(AC2) or K_(AC1).In some embodiments any of K_([C1C2]A), K_([AC2]C1), or K_([AC1]C2) isabout 1.5-fold to about 100-fold lower than any of K_(AC2) or K_(AC1);or about 2-fold to 25-fold, or about 2-fold to about 50-fold, or about1.5-fold to about 25-fold, or about 1.5-fold to about 50-fold, e.g. atleast about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold,8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold,16-fold, 17-fold, 18-fold, 19-fold or 20-fold, or up to about 100-fold,or up to about 90-fold, or up to about 80-fold, or up to about 70-fold,or up to about 60-fold, or up to about 50-fold, or up to about 40-fold,or up to about 30-fold, or up to about 20-fold, or up to about 10-foldlower. In some embodiments, any of K_([C1C2]A); K_([AC2]C1), orK_([AC1]C2) is at least about 1.5-fold lower than both of K_(AC2) orK_(AC1); or 1.5-fold to about 100-fold lower, or about 2-fold to25-fold, or about 2-fold to about 50-fold, or about 1.5-fold to about25-fold, or about 1.5-fold to about 50-fold, e.g. at least about1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold,9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold,17-fold, 18-fold, 19-fold or 20-fold, or up to about 100-fold, or up toabout 90-fold, or up to about 80-fold, or up to about 70-fold, or up toabout 60-fold, or up to about 50-fold, or up to about 40-fold, or up toabout 30-fold, or up to about 20-fold, or up to about 10-fold lower.

In some embodiments, the invention provides a negative modulatingantibody that weakens the binding of a first component (C1) to a secondcomponent (C2) of a signaling complex, said antibody characterized bythe following equilibrium dissociation constant K_(D) bindingproperties: (i) said antibody binds with an equilibrium dissociationconstant K_(D) of about 10⁻⁵M or less, e.g., 10⁻⁶M or less, or 10⁻⁷M orless, or 10⁻⁸M or less, to any one of C1, C2, or a complex comprising C1and C2 (C1C2), and (ii) any of K_(AC2) or K_(AC1) is at least about 50%(1.5-fold) lower than any of K_([C1C2]A); K_([AC2]C1); or K_([AC1]C2).In some embodiments, any of K_(AC2) or K_(AC1) is at least about1.5-fold to 100-fold lower than any of K_([C1C2]A), K_([AC2]C1); orK_([AC1]C2); or about 2-fold to 25-fold, or about 2-fold to about50-fold, or about 1.5-fold to about 25-fold, or about 1.5-fold to about50-fold, e.g. at least about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold,6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold,14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold or 20-fold, or upto about 100-fold, or up to about 90-fold, or up to about 80-fold, or upto about 70-fold, or up to about 60-fold, or up to about 50-fold, or upto about 40-fold, or up to about 30-fold, or up to about 20-fold, or upto about 10-fold lower. In some embodiments, any of K_(AC2) or K_(AC1)is at least about 1.5-fold lower than all of K_([C1C2]A); K_([AC2]C1);or K_([AC1]C2); or 1.5-fold to about 100-fold lower, or about 2-fold to25-fold, or about 2-fold to about 50-fold, or about 1.5-fold to about25-fold, or about 1.5-fold to about 50-fold, e.g. at least about1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold,9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold,17-fold, 18-fold, 19-fold or 20-fold, or up to about 100-fold, or up toabout 90-fold, or up to about 80-fold, or up to about 70-fold, or up toabout 60-fold, or up to about 50-fold, or up to about 40-fold, or up toabout 30-fold lower, or up to about 20-fold, or up to about 10-fold.

In specific embodiments, C1 and C2 are selected from the groupconsisting of insulin and insulin receptor.

In another aspect, the invention contemplates a method of preparing asterile pharmaceutical composition, comprising adding a sterilepharmaceutically acceptable diluent to an antibody of the invention.Optionally small amounts of a preservative such as a bactericidal orbacteriostatic agent are also included in the composition.

Also contemplated is a sterile composition comprising an antibody of theinvention and a sterile pharmaceutically acceptable diluent.

The invention further contemplates that the antibodies of the inventionmodulate binding between the INSR and insulin or insulin analogs orinsulin mimetics. The antibodies of the invention preferably alsoexhibit desirable biological properties, including but not limited toenhancing glucose uptake in vitro or in vivo in animal models, andpreferably the glucose uptake induced by exogenous insulin. In someembodiments, the antibodies are capable of increasing the rate or totalamount of glucose uptake, or both.

In a further aspect, the invention contemplates a method of treating adisorder associated with insulin resistance, comprising administering toa subject in need thereof a positive modulating antibody or agonistantibody of the invention in an amount effective to treat insulinresistance. In a related embodiment, the treatment enhances glucoseuptake. In a further embodiment, the enhanced glucose uptake is selectedfrom the group consisting of an increase in the rate of glucose uptake,an increase in the total amount of glucose uptake, or both. It isfurther contemplated that the treatment reduces fasting blood glucoselevels, in a subject having elevated levels of blood glucose,hyperglycemia or a disorder associated with insulin resistance, backtoward the normal range of fasting blood glucose levels. In a relatedembodiment, the fasting blood glucose is reduced by approximately 15%,20%, 25%, 30%, 35%, 40%, 45%, 50% or more compared to an untreatedsubject.

In related aspects, the treatment reduces elevated HbA1c levels, whichare a marker of elevated glucose levels over the preceding several-monthperiod, and are indicative of diabetes. In further embodiments,treatment improves impaired glucose tolerance. In one embodiment glucosetolerance is measured by glucose tolerance test (GTT).

In other embodiments, the treatment slows, reduces, or normalizes weightgain of a subject. In one embodiment, the treatment reduces or slowsweight gain by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or50% compared to an untreated subject. In some embodiments, the treatmentslows, reduces, or normalizes weight loss of a subject. In oneembodiment, the treatment reduces or slows weight loss by at least 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% compared to an untreatedsubject.

In a related aspect, it is contemplated that the antibodies orpolypeptides described herein promote or induce weight loss in asubject. In one embodiment, the invention provides a method to promoteor induce weight loss by administration of a modulating antibody,fragment thereof or polypeptide as described herein. In one embodiment,the modulating antibody is a positive modulator or partial agonist. In arelated embodiment, the modulating antibody is a negative modulator.

It certain embodiments, the treatment further results in improvement ofone, two, three or more symptoms of diabetes or insulin resistanceselected from the group consisting of dyslipidemia, elevated plasmatriglycerides, elevated HOMA-IR, elevated plasma unesterifiedcholesterol, plasma total cholesterol elevated plasma insulin(indicative of insulin resistance), low non-HDL/HDL cholesterol ratio(or low total cholesterol/HDL cholesterol ratio), and elevated plasmaleptin levels (indicative of leptin resistance).

It is further provided that the effects of treatment are also measuredusing in vitro and in vivo analysis using factors as described in theDetailed Description.

In one embodiment, the disorder associated with insulin resistance isselected from the group consisting of hyperglycemia, pre-diabetes,metabolic syndrome (also referred to as insulin resistance syndrome),Type 2 diabetes mellitus, polycystic ovary disease (PCOS), non-alcoholicfatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH),steatosis, obesity, dyslipidemia, Rabson-Mendenhall syndrome, Donohuesyndrome or Leprechaunism.

In another aspect, the invention provides a method of treating acondition or disorder associated with hyperinsulinemia, abnormalproduction and/or sensitivity to insulin that manifests as excessinsulin signaling, comprising administering to a subject in need thereofa negative modulating antibody or an antagonist antibody of theinvention in an amount effective to treat insulin overproduction and/orsensitivity. In one embodiment, the disorder associated with insulinsensitivity is selected from the group consisting of cancer, Kaposi'ssarcoma, insulinoma, diabetic renal disease, hypoglycemia,nesidioblastosis (KATP-H1 Diffuse Disease, KATP-H1 Focal Disease, or“PHHI”), GDH-H1 (Hyperinsulinism/Hyperammonaemia Syndrome (HI/HA),leucine-sensitive hypoglycemia, or diazoxide-sensitive hypoglycemia),islet cell dysregulation syndrome, idiopathic hypoglycemia of infancy,Persistent Hyperinsulinemic Hypoglycemia of Infancy (PHHI), andCongenital Hyperinsulinism, insulin overdose, hypoglycemia due to renalfailure (acute or chronic), and chronic kidney disease, e.g., type III,IV or V.

Use of any of the foregoing antibodies or polypeptides of the inventionthat modulate the insulin-INSR signaling interaction in preparation of amedicament for treatment of any of the disorders described herein isalso contemplated. Syringes, e.g., single use or pre-filled syringes,sterile sealed containers, e.g. vials, bottle, vessel, and/or kits orpackages comprising any of the foregoing antibodies or polypeptides,optionally with suitable instructions for use, are also contemplated.

Any of the foregoing antibodies or polypeptides of the invention may beconcurrently administered with any anti-diabetic agents known in the artor described herein, as adjunct therapy. Compositions comprising any ofthe foregoing antibodies or polypeptides of the invention together withother anti-diabetic agents are also contemplated.

A number of anti-diabetic agents are known in the art, including but notlimited to: 1) sulfonylureas (e.g., glimepiride, glisentide,sulfonylurea, AY31637); 2) biguanides (e.g., metformin); 3)alpha-glucosidase inhibitors (e.g., acarbose, miglitol); 4)thiazol-idinediones (e.g., troglitazone, pioglitazone, rosiglitazone,glipizide, balaglitazone, rivoglitazone, netoglitazone, troglitazone,englitazone, AD 5075, T 174, YM 268, R 102380, NC 2100, NIP 223, NIP221, MK 0767, ciglitazone, adaglitazone, CLX 0921, darglitazone, CP92768, BM 152054); 5) glucagon-like-peptides (GLP) and GLP analogs oragonists of GLP-1 receptor (e.g. exendin) or stabilizers thereof (e.g.DPP4 inhibitors, such as sitagliptin); and 6) insulin or analogues ormimetics thereof (e.g. LANTUS®).

In a related aspect, the invention provides a method of diagnosinginsulin resistance or insulin sensitivity using antibodies as describedherein. In one embodiment, the method comprises measuring levels ofinsulin or insulin receptor in a sample from a subject using an insulinreceptor antibody described herein, wherein an increased level ofinsulin or free insulin receptor, or a decreased level of membrane-boundinsulin receptor indicates the subject has or is at risk for diabetes orinsulin resistance, and optionally administering a diabetes therapeuticto said subject who has or is at risk of diabetes or insulin resistance.In another embodiment, the method comprises measuring levels of insulinreceptor in a sample from a subject using an insulin receptor antibodydescribed herein, wherein an increased level of free insulin receptor ora decreased level of membrane-bound insulin receptor indicates thesubject has or is at risk for cancer, and optionally administering acancer therapeutic to said subject.

In another aspect, the invention provides methods for identifyingantibodies that modulate the binding of insulin to the insulin receptoras described herein.

It is understood that each feature or embodiment, or combination,described herein is a non-limiting, illustrative example of any of theaspects of the invention and, as such, is meant to be combinable withany other feature or embodiment, or combination, described herein. Forexample, where features are described with language such as “oneembodiment”, “some embodiments”, “further embodiment”, “specificexemplary embodiments”, and/or “another embodiment”, each of these typesof embodiments is a non-limiting example of a feature that is intendedto be combined with any other feature, or combination of features,described herein without having to list every possible combination. Suchfeatures or combinations of features apply to any of the aspects of theinvention. Similarly, where a method describes identifying polypeptidebinding agents, such as antibodies, characterized by certain features,polypeptide binding agents characterized by those features are alsocontemplated by the invention. Where examples of values falling withinranges are disclosed, any of these examples are contemplated as possibleendpoints of a range, any and all numeric values between such endpointsare contemplated, and any and all combinations of upper and lowerendpoints are envisioned.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts representative results from an INSR receptor occupancyscreen showing test antibodies binding to IM-9 cells expressing the INSRin the presence and absence of insulin.

FIG. 2 shows representative results from a biotinylated ligand screenshowing the effects of test antibodies on insulin binding to insulinreceptor.

FIG. 3 shows results from an assay measuring the ability of testantibodies to modulate insulin dependent pIRS-1 phosphrylation.

FIG. 4 depicts results from a pIRS-1 activity assay showing binding ofrepresentative antibodies to INSR from different functional classes.FIG. 4A: Positive Modulators; FIG. 4B: Positive modulators withsignificant agonism; FIG. 4C: Non-modulators; FIG. 4D: AgonistAntibodies; FIG. 4E: Negative modulators.

FIG. 5 is a table showing insulin EC50 values for representativeantibodies from the pIRS-1 assay ranked according to EC50 ratio +Ab/−Ab.

FIG. 6 shows results of a pAKT assay for representative antibodies: FIG.6A: Positive modulator with very low agonism; FIG. 6B: Positivemodulator with agonism; FIG. 6C: Agonist antibodies; FIG. 6D: 83-7;Figure E: Insulin and background response in the absence of antibody.

FIG. 7 is a table showing agonism and mouse cross reactivity propertiesof representative test antibodies. (nd=not determined).

FIG. 8 shows pAKT assay results showing changes in sensitivity (EC50,fold-change in EC50) and cooperativity (Hillslope) of the insulin doseresponse effected by a positive modulator INSR antibody at fourdifferent concentrations. FIG. 8A shows the results graphically whileFIG. 8B shows the results in tabular form.

FIG. 9 illustrates the enhancement of insulin dependent glucose uptakeby a positive modulator antibody. ³H-2-deoxyglucose uptake in 3T3-L1cells was induced by 0.8 nM insulin in the presence of 10 ug/ml testantibody Ab001 or anti-KLH isotype control.

FIG. 10 shows blood glucose levels in 20 week old DIO mice fed a highfat diet and treated with partial agonist anti-INSR antibodies: FIG.10A: Line graph of glucose levels. FIG. 10B: Bar chart of glucose levelsshowing statistically significant reduction in blood glucose afterinjection of partial agonist anti-INSR antibody.

FIG. 11 illustrates that administration of a partial agonist anti-INSRantibody improves glycemic control in DIO mice: FIG. 11A: Glucosetolerance test timecourse; FIG. 11B: Fasting blood glucose levels; FIG.11C: Glucose tolerance test; area under curve (AUC).

FIG. 12 shows that a positive modulator anti-INSR antibody improvesinsulin sensitivity in DIO mice: FIG. 12A: Insulin tolerance testtimecourse; FIG. 12B: Fasting blood glucose levels; FIG. 12C: Insulintolerance test; area under curve (AUC).

FIG. 13 shows that a positive modulator anti-INSR antibody improvesglycemic control in DIO mice: FIG. 13A: Glucose tolerance testtimecourse; FIG. 13B: Fasting blood glucose levels; FIG. 13C: Glucosetolerance test; area under curve (AUC).

FIG. 14 demonstrates that positive modulator and partial agonistanti-INSR antibodies improve triglyceride and cholesterol levels in DIOMice. Plasma triglyceride and cholesterol levels were measured in30-week old DIO mice injected intraperitoneally (IP) with Ab001, Ab037or isotype control (10 mg/kg; * p<0.05 relative to isotype control/HFD):FIG. 14A: Plasma triglyceride levels; FIG. 14B: Plasma cholesterollevels.

FIG. 15 illustrates improvement of glycemic control in DIO mice afteradministration of positive modulator and partial agonist anti-INSRantibodies. Glycemic control measurements were observed in DIO miceinjected intraperitoneally (IP) with Ab001, Ab037, Ab083, Ab085 orisotype control (10 mg/kg; * p<0.05 relative to isotype control/HFD):FIG. 15A: Glucose tolerance test timecourse; FIG. 15B: Glucose tolerancetest; area under curve (AUC); FIG. 15C: Fasting blood glucose levels.

FIG. 16 shows positive modulator and partial agonist anti-INSRantibodies improve glycemic parameters, insulin resistance and and/ordyslipidemia in 18-week old DIO mice treated with mAb for 4 weeks.Analysis of plasma from DIO mice injected intraperitoneally (IP) withAb001, Ab037, Ab083, Ab085 or isotype control for 4 weeks (10 mg/kg; *p<0.05 relative to isotype control/HFD) was performed. FIG. 16A: Plasmaglucose levels FIG. 16B: Plasma insulin levels; FIG. 16C: HOMA-IR; FIG.16D: Plasma triglyceride levels; FIG. 16E: Plasma unesterifiedcholesterol levels; FIG. 16F: Plasma total cholesterol levels; FIG. 16G:Plasma non-HDL cholesterol levels; FIG. 16H: Plasma non-HDL/HDLcholesterol ratio.

FIG. 17 demonstrates that positive modulator and partial agonistanti-INSR antibodies reduce weight gain in DIO mice as assessed by bodyweight measurements in 18-week old DIO mice injected intraperitoneally(IP) with Ab001, Ab037, Ab083, Ab085 or isotype control for 3 weeks (10mg/kg; * p<0.05 relative to isotype control/HFD): FIG. 17A: Percentchange in body weight relative to pre-dose weight; FIG. 17B: Percentchange in body weight relative to pre-dose weight: Area under the curve.

FIG. 18 depicts positive modulator and partial agonist anti-INSRantibody-induced normalization of weight gain in db/db mice analyzed bybody weight measurements in 5 week old db/db mice injectedintraperitoneally (IP) with Ab001 (1 mg/kg or 10 mg/kg), Ab037 (10mg/kg) or isotype control (1 mg/kg or 10 mg/kg) for 14 weeks (* p<0.05relative to isotype control): FIG. 18A: Percent change in body weightrelative to pre-dose weight until day 35 of study; FIG. 18B: Percentchange in body weight relative to weight at day 35 of study; FIG. 18C:Percent change in body weight relative to pre-dose weight until day 35of study: Area under the curve; FIG. 18D: Percent change in body weightrelative to weight at day 35 of study: Area under the curve.

FIG. 19 shows that positive modulator antibody reduces fasting bloodglucose and HbA1c in db/db mice as assessed using glycemic controlmeasurements in 5 week old db/db mice injected intraperitoneally (IP)with Ab001 (1 mg/kg or 10 mg/kg), Ab037 (10 mg/kg) or isotype control (1mg/kg or 10 mg/kg) for 14 weeks (* p<0.05 relative to isotype control atsame dose): FIG. 19A: Fasting blood glucose levels; FIG. 19B: % HbA1clevels.

FIG. 20 illustrates that administration of positive modulator antibodyimproves dyslipidemia in db/db mice. Analysis of plasma from 5-week olddb/db mice injected intraperitoneally (IP) with Ab001 (1 mg/kg or 10mg/kg), Ab037 (10 mg/kg) or isotype control (1 mg/kg or 10 mg/kg) for 14weeks (* p<0.05 relative to isotype control at same dose) was carriedout. FIG. 20A: Plasma insulin levels; FIG. 20B: Plasma triglyceridelevels; FIG. 20C: Plasma unesterified cholesterol levels; FIG. 20D:Plasma total cholesterol levels; FIG. 20E: Plasma non-HDL cholesterollevels; FIG. 20F: Plasma non-HDL/HDL cholesterol ratio.

FIG. 21 shows that administration of positive modulator antibody reducesfasting blood glucose in db/db Mice. Weekly fasting blood glucoseassessment of 5-week old db/db mice injected intraperitoneally (IP) withAb001 (1 mg/kg or 10 mg/kg), Ab037 (10 mg/kg) or isotype control (1mg/kg or 10 mg/kg) for 14 weeks (* p<0.05 for Ab085 relative to isotypecontrol at same dose).

FIG. 22 illustrates that positive modulator antibodies improve insulinresistance in db/db mice as assessed by analysis of plasma from 5-weekold db/db mice injected intraperitoneally (IP) with Ab001, Ab037, Ab083,Ab085 or isotype control for 4 weeks (10 mg/kg; * p<0.05 relative toisotype control at same dose) and shows Homeostatic model assessment forInsulin Resistance (HOMA-IR) after 4 weeks of dosing.

FIG. 23 illustrates that positive modulator and partial agonistanti-INSR antibodies improve glycemic control in MLDS/HFD mice. Glycemiccontrol measurements were taken from 10-week old MLDS/HFD mice injectedintraperitoneally (IP) with Ab001, Ab037 or isotype control (10 mg/kg; *p<0.05 relative to isotype control): FIG. 23A: Glucose tolerance testtimecourse; FIG. 23B: Glucose tolerance test; area under curve (AUC);FIG. 23C: Fasting blood glucose levels.

FIG. 24 shows administration of a partial agonist antibody reduces fedblood glucose and HbA1c in MLDS/HFD mice. Glycemic control measurementswere taken in 10-week old MLDS/HFD mice injected intraperitoneally (IP)with Ab001, Ab037 or isotype control for 6 weeks (10 mg/kg; * p<0.05relative to isotype control): FIG. 24A: Fed blood glucose levels; FIG.24B: % HbA1c levels.

FIG. 25 shows positive modulator and/or partial agonist anti-INSRantibodies partially correct insulin, leptin and non-HDL/HDL cholesterollevels in MLDS/HFD mice. Plasma cholesterol, insulin and leptin levelswere measured in 10-week old MLDS/HFD mice injected intraperitoneally(IP) with Ab001, Ab037 or isotype control for 6 weeks (10 mg/kg; *p<0.05 relative to isotype control): FIG. 25A: Plasma non-HDL/HDLcholesterol ratio; FIG. 25B: Plasma insulin levels; FIG. 25C: Plasmaleptin levels.

FIG. 26 illustrates that positive modulator and partial agonistanti-INSR antibodies do not affect body weight in MLDS/HFD mice. Bodyweight measurements were taken in 10-week old MLDS/HFD mice injectedintraperitoneally (IP) with Ab001, Ab037 or isotype control for 6 weeks(10 mg/kg) and results expressed as percent change in body weightrelative to pre-dose weight.

FIG. 27 illustrates that positive modulator and partial agonistanti-INSR antibodies improve glycemic control in MLDS/HFD mice. Aglucose tolerance test (GTT) was carried out in 10-week old MLDS/HFDmice injected intraperitoneally (IP) with Ab001, Ab083, Ab085, Ab037 orisotype control for 3 weeks (10 mg/kg; * p<0.05 relative to isotypecontrol). FIG. 27A: Glucose tolerance test timecourse; FIG. 27B: Glucosetolerance test; area under curve (AUC).

FIG. 28 shows that positive modulator and partial agonist anti-INSRantibodies improve glycemic control in MLDS/HFD mice as determined by aweekly fasting blood glucose assessment of 10-week old MLDS/HFD miceinjected intraperitoneally (IP) with Ab001, Ab083, Ab085, Ab037 orisotype control for 6 weeks (10 mg/kg; * p<0.05 for Ab083 and Ab037relative to isotype control).

FIG. 29 illustrates that positive modulator and partial agonistanti-INSR antibodies improve dyslipidemia in MLDS/HFD mice. Analysis ofplasma from of 10-week old MLDS/HFD mice injected intraperitoneally (IP)with Ab001, Ab083, Ab085, Ab037 or isotype control for 6 weeks (10mg/kg; * p<0.05 relative to isotype control) was carried out. FIG. 29A:Plasma triglyceride levels; FIG. 29B: Plasma free fatty acid levels;FIG. 29C: Plasma unesterified cholesterol levels; FIG. 29D: Plasma totalcholesterol levels; FIG. 29E: Plasma non-HDL cholesterol levels; FIG.29F: Plasma non-HDL/HDL cholesterol ratio.

FIG. 30 shows that positive modulator and partial agonist anti-INSRantibodies improve glycemic control (HbA1c) in MLDS/HFD mice asdetermined by a blood HbA1c evaluation in 10-week old MLDS/HFD miceinjected intraperitoneally (IP) with Ab001, Ab083, Ab085, Ab037 orisotype control for 6 weeks (10 mg/kg; * p<0.05 for Ab083 and Ab037relative to isotype control).

FIG. 31 shows that positive modulator and partial agonist anti-INSRantibodies generally do not affect body weight in MLDS/HFD mice asdetermined using body weight measurements in 10-week old MLDS/HFD miceinjected intraperitoneally (IP) with Ab001, Ab083, Ab085, Ab037 orisotype control for 6 weeks (10 mg/kg).

FIG. 32 shows that positive modulator and partial agonist anti-INSRantibodies augment insulin signaling in vivo. Ten week-old C56BL/6 malemice were injected with Ab083, Ab085, Ab037 or isotype control (10mg/kg) for 24 hours, and effects on liver (FIG. 32A) and muscle (FIG.32B) INSR tyrosine phosphorylation were determined by ELISA after aninsulin bolus.

FIG. 33 is a table showing the binding characteristics of INSR-specificantibodies reformatted with an IgG2 constant region.

FIG. 34A illustrates the dose response from a partial allosteric agonistanti-INSR antibody in comparison to the dose response to the endogenousligand and FIG. 34B illustrates activation by ligand in the presence orabsence of the allosteric agonist antibody.

FIG. 35A shows the dose response from a positive modulator antibody incomparison to the dose response to the endogenous ligand and FIG. 35Bshows the dose response of an endogenous ligand in the presence andabsence of a positive modulator antibody.

FIG. 36 illustrates the activation parameters for a set of partialallosteric agonists alone relative to the endogenous ligand insulin.Data obtained from measurements of percent Akt phosphorylation atSer473.

FIG. 37 illustrates the activation properties of insulin in the presenceof 10 ug/ml partial allosteric agonist antibodies relative to themaximal response to the endogenous ligand in the presence of a negativecontrol antibody. Data obtained from measurements of percent Aktphosphorylation at Ser473.

FIGS. 38-40 depict pAkt activation by antibodies in the absence ofinsulin or in the presence of a sub-maximal concentration of insulin forparental CHO-K1 cells, CHO-K1 cells expressing human insulin receptorand CHO-K1 cells expressing mouse insulin receptor. FIGS. 38A-C show theeffects of sensitizer Abs (Ab077, Ab078, Ab085) with little or noagonism of pAkt activity (<10% pAkt activation in the absence of insulinwith 50 ug/ml antibody). FIGS. 39A-C show the effects of sensitizer Abs(Ab001, Ab079, Ab083) with weak to moderate agonism of pAkt activity(10-20% pAkt activation in the absence of insulin with 50 ug/mlantibody). FIG. 40 illustrates the effects of a sensitizer Ab (Ab080)with moderate agonism of pAkt activity (>20% pAkt activation in theabsence of insulin with 50 ug/ml antibody).

FIG. 41A and FIG. 41C shows insulin dependent pAkt activation in CHOcells expressing the human and FIG. 41B shows mouse INSR in the presenceof fixed concentrations of sensitizing anti-INSR antibodies.

FIG. 42A demonstrates pAkt activation in CHO cells expressing the humanand FIG. 42B demonstrates mouse INSR by partial allosteric agonistantibodies in the absence of insulin compared to insulin-alone.

FIG. 43 depicts the results of insulin dependent pAkt activation in CHOcells expressing the human INSR in the presence of fixed concentrationsof partial allosteric agonist antibodies.

FIG. 44 shows pAKT assay results for antibody 83-7 and Ab001 on CHOK1cells expressing: FIG. 44A: human INSR, or; FIG. 44B: mouse INSR.

FIG. 45 shows free insulin percentage plotted against estimated insulinreceptor concentration. The insulin level was fixed at 50 pM and theantibody concentration was 10 ug/mL (67 nM) for all clones except Ab078which was tested at 25 ug/mL (167 nM). Curves shown are the non-linearregression Prism fit used to calculate EC50.

FIG. 46 shows free insulin percentage plotted against estimated insulinreceptor concentration. The insulin level was fixed at 50 pM and theantibody concentration was 10 ug/mL (67 nM) for all clones. Curves shownare the non-linear regression Prism fit used to calculate EC50.

FIG. 47 shows that TNFα-induces desensitization of insulin-mediatedfatty acid uptake in 3T3-L1 adipocytes in the presence of anti-INSRantibody Ab085.

FIG. 48 and FIG. 49 illustrate the effects of purified positivemodulator anti-INSR antibodies Ab001, Ab037, Ab077, Ab079, AB080, Ab083on human INSR (FIG. 48) and mouse INSR (FIG. 49). as measured in thepAKT assay.

FIGS. 50 and 51 demonstrates the relative % pAKT of purified agonistantibodies Ab037, Ab030, Ab053 and Ab062 on human INSR (FIG. 50) andmouse INSR (FIG. 51).

FIG. 52 demonstrates that the purified anti-INSR antibodies Ab030,Ab037, Ab053, Ab001, Ab079, AB080 and Ab083 are capable of inducing AKTphosphorylation (relative % pAKT) after activation of monkey INSR.

FIG. 53 shows the relative % pAKT of negative modulator antibodiesAb061, Ab070 and Ab081 measured in CHOK1 cells expressing human INSR.

FIG. 54 is a table showing cross-reactivity of insulin receptorantibodies, and illustrates that certain antibodies that bind to thehuman insulin receptor also bind to the rabbit and the cynomolgousinsulin receptor and that this binding was modulated by the presence ofinsulin.

DETAILED DESCRIPTION

The invention provides antibodies specific for the insulin receptor(INSR) or the insulin receptor-insulin complex and uses thereof in thetreatment of disorders related to aberrant glucose levels, e.g.hyperglycemia or hypoglycemia, aberrant insulin levels or aberrantinsulin sensitivity, e.g. disorders of insulin resistance or disordersof insulin sensitivity. These antibodies can induce either a positive ornegative effect on the cellular response in the INSR by altering thekinetic rate constants for assembly and dissociation of INSR-INSsignaling complex components or by other mechanisms including alteringthe structural state of the signaling complex, e.g., by binding to atransition state and accelerating the activation of signaling.

Modulation of a signaling complex can result in an increase or decreasein sensitivity to signal input and concomitant increases or decreases insignal transduction. Administration of modulator antibodies increases ordecreases the sensitivity of the cellular pathway and/or absolute levelsof the cellular response. The modulators of the invention, depending ontheir properties, can function as a modulator, potentiator, regulator,effector or sensitizer.

Many antibody drugs act to block signaling pathways by binding to eithera cell-surface receptor or its cognate ligand and eliminating theability of the ligand to bind to and activate the receptor. Suchblocking drugs mediate their effect stoichiometrically by preventing theformation of receptor-ligand complex.

Successful treatment of some diseases may require attenuation ratherthan complete inhibition of signaling pathways to restore a normalphysiological state with acceptable side-effect profiles. The antibodiesprovided by the invention are expected to provide such advantages.

Other therapeutic drugs affect cellular signaling pathways by binding toa cell-surface receptor and altering the activity of the receptor. Suchdirect agonist drugs may mediate their effects by mimicking the naturalactivity of the ligand and thus have inherent activity, i.e., they donot require the presence of ligand to mediate their effects. Furthertherapeutic drugs affect cellular signaling pathways by binding to aligand. Such indirect agonist drugs may mediate their effects byaltering ligand stability or valency.

Biological processes are generally regulated in a continuous rather thanbinary manner, and thus in many cases modulation of pathway activity maybe a more appropriate therapeutic strategy than complete pathwayblockade or stimulation. Performing functional, cell-based screens formodulation of pathway activity, rather than for complete pathwayblockade or stimulation, is laborious and may not readily be performedin a high throughput manner, since such screens generally require aknown concentration of test compound and may be sensitive to anyimpurities in the test compound preparation. In particular, the abilityto perform high throughput functional, cell-based screens for modulationof pathway activity is restricted for cell-impermeable molecules whichare unable to enter the intracellular environment, and especially forrecombinant biological molecules which may have different expressionlevels, degrees of purity and stability in the production system used.In addition, some binding interactions may have no signaling output tomeasure in a functional screen (e.g. in the case of decoy receptors,decoy substrates, or inactive forms of a target) making it difficult toidentify agents that perturb these interactions.

The present invention overcomes these disadvantages and provides a meansfor identifying positive and negative modulators of the INSR activityand desired potency of drug in a high throughput manner. The presentinvention also provides positive and negative modulators of the INSRactivity with a desired range of modulation of activity, and providesdata showing that these modulators exhibit the desired biological effectof altering glucose uptake.

Definitions

The term “compound” refers to any chemical compound, organic orinorganic, endogenous or exogenous, including, without limitation,polypeptides, proteins, peptides, small molecules, nucleic acids (e.g.DNA and RNA), carbohydrates, lipids, fatty acids, steroids, purines,pyrimidines, peptidomimetics, polyketides and derivatives, structuralanalogs or combinations thereof. “Endogenous” means naturally occurringin a mammal, while “exogenous” means not naturally occurring in themammal, e.g. an administered foreign compound.

The term “polypeptide binding agent” refers to a polypeptide that iscapable of specifically binding an antigen, e.g. a target or itssignaling partner, or that is capable of binding an antigen with ameasurable binding affinity. Examples of polypeptide binding agentsinclude antibodies, peptibodies, polypeptides and peptides, optionallyconjugated to other peptide moieties or non-peptidic moieties. Antigensto which a polypeptide binding agent may bind include any proteinaceousor non-proteinaceous molecule that is capable of eliciting an antibodyresponse, or that is capable of binding to a polypeptide binding agentwith detectable binding affinity greater than non-specific binding. Theantigen to which a modulating polypeptide binding agent binds mayinclude a target, a signaling partner of a target, and/or a complexcomprising the target and its signaling partner.

The term “antibody” is used in the broadest sense and includes fullyassembled antibodies, tetrameric antibodies, monoclonal antibodies,polyclonal antibodies, multispecific antibodies (e.g., bispecificantibodies), antibody fragments that can bind an antigen (e.g., Fab′,F′(ab)2, Fv, single chain antibodies, diabodies), and recombinantpeptides comprising the forgoing as long as they exhibit the desiredbiological activity. An “immunoglobulin” or “tetrameric antibody” is atetrameric glycoprotein that consists of two heavy chains and two lightchains, each comprising a variable region and a constant region.Antigen-binding portions may be produced by recombinant DNA techniquesor by enzymatic or chemical cleavage of intact antibodies. Antibodyfragments or antigen-binding portions include, inter alia, Fab, Fab′,F(ab′)2, Fv, domain antibody (dAb), complementarity determining region(CDR) fragments, CDR-grafted antibodies, single-chain antibodies (scFv),single chain antibody fragments, chimeric antibodies, diabodies,triabodies, tetrabodies, minibody, linear antibody; chelatingrecombinant antibody, a tribody or bibody, an intrabody, a nanobody, asmall modular immunopharmaceutical (SMIP), a antigen-binding-domainimmunoglobulin fusion protein, a camelized antibody, a VHH containingantibody, or a variant or a derivative thereof, and polypeptides thatcontain at least a portion of an immunoglobulin that is sufficient toconfer specific antigen binding to the polypeptide, such as one, two,three, four, five or six CDR sequences, as long as the antibody retainsthe desired biological activity.

“Monoclonal antibody” refers to an antibody obtained from a populationof substantially homogeneous antibodies, i.e., the individual antibodiescomprising the population are identical except for possible naturallyoccurring mutations that may be present in minor amounts.

“Antibody variant” as used herein refers to an antibody polypeptidesequence that contains at least one amino acid substitution, deletion,or insertion in the variable region of the natural antibody variableregion domains. Variants may be substantially homologous orsubstantially identical to the unmodified antibody.

A “chimeric antibody,” as used herein, refers to an antibody containingsequence derived from two different antibodies (see, e.g., U.S. Pat. No.4,816,567) which typically originate from different species. Mosttypically, chimeric antibodies comprise human and rodent antibodyfragments, generally human constant and mouse variable regions.

A “neutralizing antibody” is an antibody molecule which is able toeliminate or significantly reduce a biological function of an antigen towhich it binds. Accordingly, a “neutralizing” antibody is capable ofeliminating or significantly reducing a biological function, such asenzyme activity, ligand binding, or intracellular signaling.

An “isolated” antibody is one that has been identified and separated andrecovered from a component of its natural environment. Contaminantcomponents of its natural environment are materials that would interferewith diagnostic or therapeutic uses for the antibody, and may includeenzymes, hormones, and other proteinaceous or non-proteinaceous solutes.In preferred embodiments, the antibody will be purified (1) to greaterthan 95% by weight of antibody as determined by the Lowry method, andmost preferably more than 99% by weight, (2) to a degree sufficient toobtain at least 15 residues of N-terminal or internal amino acidsequence by use of a spinning cup sequenator, or (3) to homogeneity bySDS-PAGE under reducing or nonreducing conditions using Coomassie blueor, preferably, silver stain. Isolated antibody includes the antibody insitu within recombinant cells since at least one component of theantibody's natural environment will not be present. Ordinarily, however,isolated antibody will be prepared by at least one purification step.

“Heavy chain variable region” as used herein refers to the region of theantibody molecule comprising at least one complementarity determiningregion (CDR) of said antibody heavy chain variable domain. The heavychain variable region may contain one, two, or three CDR of saidantibody heavy chain.

“Light chain variable region” as used herein refers to the region of anantibody molecule, comprising at least one complementarity determiningregion (CDR) of said antibody light chain variable domain. The lightchain variable region may contain one, two, or three CDR of saidantibody light chain, which may be either a kappa or lambda light chaindepending on the antibody.

As used herein, an antibody that “specifically binds” is “antigenspecific”, is “specific for” antigen target or is “immunoreactive” withan antigen refers to an antibody or polypeptide binding agent of theinvention that binds an antigen with greater affinity than otherantigens of similar sequence. In one aspect, the polypeptide bindingagents of the invention, or fragments, variants, or derivatives thereof,will bind with a greater affinity to human antigen as compared to itsbinding affinity to similar antigens of other, i.e., non-human, species,but polypeptide binding agents that recognize and bind orthologs of thetarget are within the scope of the invention.

For example, a polypeptide binding agent that is an antibody or fragmentthereof “specific for” its cognate antigen indicates that the variableregions of the antibodies recognize and bind the desired antigen with adetectable preference (e.g., where the desired antigen is a polypeptide,the variable regions of the antibodies are able to distinguish theantigen polypeptide from other known polypeptides of the same family, byvirtue of measurable differences in binding affinity, despite thepossible existence of localized sequence identity, homology, orsimilarity between family members). It will be understood that specificantibodies may also interact with other proteins (for example, S. aureusprotein A or other antibodies in ELISA techniques) through interactionswith sequences outside the variable region of the antibodies, and inparticular, in the constant region of the molecule. Screening assays todetermine binding specificity of a polypeptide binding agent, e.g.antibody, for use in the methods of the invention are well known androutinely practiced in the art. For a comprehensive discussion of suchassays, see Harlow et al. (Eds), Antibodies A Laboratory Manual; ColdSpring Harbor Laboratory; Cold Spring Harbor, N.Y. (1988), Chapter 6.Antibodies for use in the invention can be produced using any methodknown in the art.

The term “epitope” refers to that portion of any molecule capable ofbeing recognized by and bound by a selective binding agent at one ormore of the antigen binding regions. Epitopes usually consist ofchemically active surface groupings of molecules, such as, amino acidsor carbohydrate side chains, and have specific three-dimensionalstructural characteristics as well as specific charge characteristics.Epitopes as used herein may be contiguous or non-contiguous.

The term “derivative” when used in connection with polypeptide bindingagents and polypeptides of the invention refers to polypeptideschemically modified by such techniques as ubiquitination, conjugation totherapeutic or diagnostic agents, labeling (e.g., with radionuclides orvarious enzymes), covalent polymer attachment such as pegylation(derivatization with polyethylene glycol) and insertion or substitutionby chemical synthesis of amino acids such as ornithine, which do notnormally occur in human proteins. Derivatives retain the bindingproperties of underivatized molecules of the invention.

“Detectable moiety” or a “label” refers to a composition detectable byspectroscopic, photochemical, biochemical, immunochemical, or chemicalmeans. For example, useful labels include 32P, 35S, fluorescent dyes,electron-dense reagents, enzymes (e.g., as commonly used in an ELISA),biotin-streptavadin, dioxigenin, haptens and proteins for which antiseraor monoclonal antibodies are available, or nucleic acid molecules with asequence complementary to another labeled nucleic acid molecule. Thedetectable moiety often generates a measurable signal, such as aradioactive, chromogenic, or fluorescent signal, that can be used toquantitate the amount of bound detectable moiety in a sample.

“Peptides” or “oligopeptides” are short amino acid sequences, typicallybetween 3 and 100 amino acid residues in length and encompass naturallyoccurring amino acid residues and non-naturally occurring analogs ofresidues which may be used singly or in combination with naturallyoccurring amino acid residues in order to give the peptide a particularconformational specificity or a particular biological activity, such asresistance to proteolysis. Peptides include repeats of peptide sequencesand may include 2, 3, 4, 5, 6, 7, 8, 9, 10 or more copies of an aminoacid sequence arranged head-to-tail or head-to-head. Peptides may beconjugated to non-peptidic moieties, e.g. [expand]. Peptides includedimers, trimers or higher order multimers, e.g. formed throughconjugation to other polymeric or non-polymeric moieties, such as PEG.

“Polypeptides” are longer amino acid sequences, typically 100 or moreamino acid residues in length, and encompass naturally occurring aminoacid residues and non-naturally occurring analogs of residues which maybe used singly or in combination with naturally occurring amino acidresidues in order to give the polypeptide a particular conformationalspecificity or a particular biological activity, such as resistance toproteolysis.

As used herein, a “peptibody” is a fusion polypeptide comprising one ormore peptides fused to all or a portion of an immunoglobulin (Ig)constant region. See, e.g., U.S. Pat. No. 6,660,843. The peptide may beany naturally occurring or recombinantly prepared or chemicallysynthesized peptide that binds to the antigen. The peptide may berepeated and may include 2, 3, 4, 5, 6, 7, 8, 9, 10 or more copies of anamino acid sequence arranged head-to-tail or head-to-head. The portionof the Ig constant region may include at least one constant regiondomain (e.g., CH1, CH2, CH3, and/or CH4), multiple domains (e.g., CH2with CH3), multiple copies of domains (e.g., CH2-CH2), any fragment of aconstant domain that retains the desired activity, e.g. the salvagereceptor epitope responsible for the prolonged half-life ofimmunoglobulins in circulation, or any combinations thereof.

A “small” molecule or “small” organic molecule is defined herein as anon-polymeric organic chemical compound having a molecular weight ofabout 1000 Daltons or less.

As used herein, a “signaling complex” is an assembly of proteins and/orendogenous or exogenous compounds that mediate the transduction of acellular signal. Examples of a signaling complex include, but are notlimited to, a ligand bound to a membrane bound receptor, an enzyme boundto a substrate or any cellular molecules that associate to propagatebiochemical reactions that are involved in a signal cascade. Signalingcomplexes can also include coreceptors, cofactors, scaffold proteins,allosteric modulators and numerous other types of proteins and moleculesthat are involved in cellular signal transduction. Signaling complexescan be formed transiently or can be long lived. The molecularconstituents or components of a signaling complex can vary over time andcan be dependent on activation state of each component and the cellularenvironment. Signaling complexes can undergo chemical modification andregulation that can induce a spectrum of effects on the complexincluding subtle changes in transduction activity, complete inactivationand constitutive activation or both positive and negative modulation.

The term “therapeutically effective amount” is used herein to indicatethe amount of target-specific composition of the invention that iseffective to ameliorate or lessen symptoms or signs of diseaseassociated with abnormal (e.g. abnormally high or abnormally low)signaling of the signaling complex.

As used herein “binding” is the physical association between two or moredistinct molecular entities that results from a specific network ofnon-covalent interactions consisting of one or more of the weak forcesincluding hydrogen bonds, Van der Waals, ion-dipole and hydrophobicinteractions and the strong force ionic bonds. The level or degree ofbinding may be measured in terms of affinity. Affinity is a measure ofthe strength of the binding interaction between two or more distinctmolecular entities that can be defined by equilibrium binding constantsor kinetic binding rate parameters. Examples of suitable constants orparameters and their measurement units are well known in the art andinclude but are not limited to equilibrium association constant (K_(A)),e.g. about 10⁵M⁻¹ or higher, about 10⁶M⁻¹ or higher, about 10⁷M⁻¹ orhigher, about 10⁸M⁻¹ or higher, about 10⁹M⁻¹ or higher, about 10¹⁰M⁻¹ orhigher, about 10¹¹M⁻¹ or higher or about 10¹²M⁻¹ or higher; equilibriumdissociation constant (K_(D)), e.g., about 10⁻⁵M or less, or about 10⁻⁶Mor less, or about 10⁻⁷M or less, or about 10⁻⁸M or less, or about 10⁻⁹Mor less, or about 10⁻¹⁰M or less, or about 10⁻¹¹M or less, or about10⁻¹²M or less; on-rate (e.g., sec⁻¹, mol⁻¹) and off-rate (e.g.,sec-¹)). In the case of K_(A), higher values mean “stronger” or“strengthened” binding affinity while in the case of K_(D), lower valuesmean “stronger” or “strengthened” binding affinity. As used herein, a“strengthened” binding rate parameter means increased residency time,stronger association or weaker dissociation. As used herein, a“weakened” binding rate parameter means decreased residency time, weakerassociation or stronger dissociation. In the case of on-rate, highervalues mean faster or more frequent association and thus generallyresult in strengthened binding affinity. In the case of off-rate, lowervalues generally mean slower dissociation and thus generally result instronger binding affinity. However, it is the ratio of the on-rate andoff-rate that indicates binding affinity, as explained in further detaillater.

Affinity between two compounds, e.g., between an antibody and anantigen, or between first and second components of a signaling complex,may be measured directly or indirectly. Indirect measurement of affinitymay be performed using surrogate properties that are indicative of,and/or proportional to, affinity. Such surrogate properties include: thequantity or level of binding of a first component to a second componentof a signaling complex, or a biophysical characteristic of the firstcomponent or the second component that is predictive of or correlated tothe apparent binding affinity of the first component for the secondcomponent. Specific examples include measuring the quantity or level ofbinding of first component to a second component at a subsaturatingconcentration of either the first or the second component. Otherbiophysical characteristics that can be measured include, but are notlimited to, the net molecular charge, rotational activity, diffusionrate, melting temperature, electrostatic steering, or conformation ofone or both of the first and second components. Yet other biophysicalcharacteristics that can be measured include determining stability of abinding interaction to the impact of varying temperature, pH, or ionicstrength.

Measured affinity is dependent on the exact conditions used to make themeasurement including, among many other factors, concentration ofbinding components, assay setup, valence of binding components, buffercomposition, pH, ionic strength and temperature as well as additionalcomponents added to the binding reaction such as allosteric modulatorsand regulators. Quantitative and qualitative methods may be used tomeasure both the absolute and relative strength of binding interactions.

Apparent affinity is a measure of the strength of the bindinginteraction between two or more distinct molecular entities underconditions where the affinity is altered by conditions or components inthe binding reaction such as allosteric modulators, inhibitors, bindingcomponent valence etc.

As used herein a “subsaturating concentration” is a concentration of oneor more components in a binding reaction that is significantly below thebinding affinity K_(D) and/or a concentration of one component in abinding reaction that is less than is required to occupy all of thebinding sites of the other component(s). Under subsaturating conditionsa significant percentage of one of the binding components in the bindingreaction has available binding sites.

As used herein a “biophysical assay” is any method that measures, in anabsolute or relative fashion, the binding, association, dissociation,binding affinity, binding level, or binding rate parameters between atleast two compounds. Biophysical assays are generally performed in vitroand may be conducted with purified binding components, unpurifiedcomponents, cell associated components as well as a combination ofpurified and cell associated components.

An “agonist” is a term used to describe a type of ligand or drug thatbinds and activates signaling of a receptor. The ability to alter theactivity of a receptor, also known as the agonist's efficacy, is aproperty that distinguishes it from antagonists, a type of receptorligand which also binds a receptor but which does not activate signalingof the receptor. The efficacy of an agonist may be positive, causing anincrease in the receptor's activity, or negative causing a decrease inthe receptor's activity. Full agonists bind and activate a receptor,displaying full efficacy at that receptor. Partial agonists also bindand activate a given receptor, but have only partial efficacy at thereceptor relative to a full agonist. An inverse agonist is an agentwhich binds to the same receptor binding-site as an agonist for thatreceptor and reverses constitutive activity of receptors. Inverseagonists exert the opposite pharmacological effect of a receptoragonist. A co-agonist works with other co-agonists to produce thedesired effect together.

Competitive, or orthosteric, agonists reversibly bind to receptors atthe same binding site (active site) as the ligand, thereby competingwith ligand for the same binding site on the receptor.

In a different aspect, antibodies disclosed herein act as allostericagonists. They bind to a portion of INSR that is distinct from theactive insulin-binding site, and do not appreciably change the bindingaffinity of insulin and INSR by more than 2-fold. They also do notappreciably affect the EC50 of insulin activation of INSR, e.g. alterEC50 by less than 2-fold. Such antibodies constitutively activate INSRwith a maximal agonist response that is 80% or less of the maximalagonist response of insulin, for example 15%-80%, 20-60%, 20%-40% or15%-30%. In certain embodiments, the antibodies constitutively activateINSR with a maximal agonist response that is at least about 15%, 20%,25%, 30%, 35%, 40%; and up to 45%, 50%, 55%, 60%, 65%, 70%, 75% or 80%of the maximal agonist response of insulin. It is understood that anycombination of any of these range endpoints is contemplated withouthaving to recite each possible combination. In some embodiments, maximalagonist response is measured by Akt assay. In further embodiments, theinvention provides an allosteric agonist antibody that binds to insulinreceptor with an affinity of 10⁻⁵, 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹, 10⁻¹⁰, 10⁻¹¹M or less M. Without being bound by a theory of the invention, the weakagonist activity of allosteric agonists serves to mimic the effect ofnatural basal insulin secretion levels, while permitting exogenouslyadministered insulin to have its normal glucose-lowering effect. Incertain embodiments, an allosteric agonist is a partial allostericagonist. An antagonist blocks a receptor from activation by agonists. Aselective agonist is selective for one certain type of receptor. It canadditionally be of any of the aforementioned types.

The potency of an agonist is usually defined by the inverse of its EC50value. This can be calculated for a given agonist by determining theconcentration of agonist needed to elicit half of the maximum biologicalresponse of the agonist. The lower the EC50, the greater the potency ofthe agonist.

A receptor “antagonist” is a type of receptor ligand or drug that doesnot provoke a biological response itself upon binding to a receptor, butblocks or dampens agonist-mediated responses. Antagonists may haveaffinity but no efficacy for their cognate receptors, and binding willdisrupt the interaction and inhibit the function of an agonist orinverse agonist at receptors. Antagonists mediate their effects bybinding to the active site or to allosteric sites on receptors, or theymay interact at unique binding sites not normally involved in thebiological regulation of the receptor's activity. Antagonist activitymay be reversible or irreversible depending on the longevity of theantagonist-receptor complex, which, in turn, depends on the nature ofantagonist receptor binding. The majority of antagonists achieve theirpotency by competing with endogenous ligands or substrates atstructurally-defined binding sites on receptors.

Antagonists display no efficacy to activate the receptors they bind.Once bound, however, antagonists may inhibit the function of agonists,inverse agonists and partial agonists. In functional antagonist assays,a dose-response curve measures the effect of the ability of a range ofconcentrations of antagonists to reverse the activity of an agonist. Thepotency of an antagonist is usually defined by its IC50 value. This canbe calculated for a given antagonist by determining the concentration ofantagonist needed to elicit half inhibition of the maximum biologicalresponse of an agonist. The lower the IC50, the greater the potency ofthe antagonist.

Competitive, or orthosteric, antagonists reversibly bind to receptors atthe same binding site (active site) as the ligand or agonist, butwithout activating the receptor, thereby competing with agonist for thesame binding site on the receptor. Non-competitive, or allosteric,antagonists bind to a separate binding site from the agonist, exertingtheir action to that receptor via that separate binding site. Thus, theydo not compete with agonists for binding. Uncompetitive antagonistsdiffer from non-competitive antagonists in that they require receptoractivation by an agonist before they can bind to a separate allostericbinding site.

“Insulin resistance” describes a condition in which physiologicalamounts of insulin are inadequate to produce a normal insulin responsefrom cells or tissues.

“Insulin sensitizer” is a compound or drug that increases cell- ortissue-sensitivity to insulin resulting in greater levels of glucoseuptake for a given subsaturating concentration of insulin.

Advantages

The present invention relates to the discovery that it is possible todevelop therapeutic agents that modulate the insulin-INSR signalingcomplex by binding to extracellular regions of the INSR. Novel selectionand screening methods are employed to identify, for example,insulin-sensitizers that target extracellular regions of the INSR andpotentiate insulin action. In particular, some of the antibodiesidentified herein are non-agonistic antibodies which bind toextracellular regions of the INSR and positively or negatively modulatethe insulin-INSR signaling complex.

The present invention encompasses insulin-INSR signaling complexmodulators that offer unique advantages over existing therapies. Theyact at the level of the INSR, which should allow induction of the entirerange of actions of insulin while minimizing unwanted side effects.Avoiding INSR agonism should reduce the risk of functional hypoglycemia.Additionally, more precise control of glucose levels might be achieved.Thus, when blood glucose levels increase, leading to elevation ofinsulin levels, such a modulator would have a greater effect. Targetingthe extracellular region of the INSR allows for the use of biologicalmolecules as insulin-INSR signaling complex modulators that modulate theeffect of endogenous or exogenous insulin, insulin analogs or insulinmimetics; these may have advantages such as improved half-life, reduceddosage or frequency of dosage, reduced toxicity and greater ease ofmanufacture. The present invention encompasses insulin-INSR signalingcomplex modulators that are expected to reduce peripheral insulinresistance and improve glycemic control. The sensitizing effect of themodulators should allow for improved levels of glucose uptake byperipheral tissues in patients whose insulin levels are not high enoughto stimulate adequate glucose uptake in the absence of exogenous insulintherapy. Thus, administration of the antibodies of the invention may beused in the early stages of insulin resistance in place of other drugs,or as adjunct therapy to other anti-diabetic agents. When administeredas an adjunct therapy to other anti-diabetic agents, the antibodies ofthe invention may reduce the total daily amount of anti-diabetic agentrequired to maintain blood glucose levels closer to normal range, or mayreduce the frequency of dosing of the anti-diabetic agent, or mayachieve improved glycemic control with the same dose and/or frequency,e.g. with fewer episodes of hyperglycemia or a reduced level of maximalhyperglycemia (reduction in the highest aberrant glucose levelobserved).

The Insulin Receptor (INSR)

The INSR is a tyrosine kinase receptor found in organisms as primitiveas cnidarians and insects. In higher organisms it is essential forglucose homeostasis. Mouse knockout studies have also shown the INSR tobe important in adipogenesis, neovascularization, the regulation ofhepatic glucose synthesis and glucose-induced pancreatic insulinsecretion (Kitamura et al, Ann. Rev. Physiol., 65: 313-332 2003). INSRsignaling is also important in the brain, where it is involved in theregulation of food intake, peripheral fat deposition and thereproductive endocrine axis as well as in learning and memory (Wada etal, J. Pharmacol. Sci. 99: 128-143, 2005). Dysfunctional INSR signalinghas been implicated in diseases including type I and type II diabetes,dementia and cancer.

The domains of the closely related insulin-like growth factor receptor(IGFR-1) exhibit high (47-67%) amino acid identity with the INSR. Whilesimilar in structure, IGF-IR and INSR serve different physiologicalfunctions. IGF-IR is expressed in almost all normal adult tissue exceptfor liver, which is itself the major site of IGF-I production. INSR isprimarily involved in metabolic functions whereas IGF-IR mediates growthand differentiation (Adams et al, Cell. Mol. Life Sci. 57: 1050-1093,2000).

INSR exists as two splice variant isoforms, INSR-A and INSR-B, whichrespectively lack or contain the 12 amino acids coded by exon 11. Thelonger variant, INSR-B, is the isoform responsible for signalingmetabolic responses. In contrast, INSR-A signals predominantly mitogenicresponses, is the preferentially expressed isoform in several cancers(Denley et al., Horm. Metab. Res. 35: 778-785, 2003) and is capable ofbinding insulin-like growth factor 2 (IGF-II) with high affinity (Denleyet al, Mol. Endocrinol. 18: 2502-2512, 2004).

The mature human INSR is a homodimer comprising two a subunits and two βsubunits (chains). The α and β chains are encoded by a single gene andarise from the post-translational cleavage of a 1370 amino acidprecursor at a furin cleavage site located at residues 720-723. Theα-chain and 194 residues of the β-chain comprise the extracellularprotion of the INSR. There is a single transmembrane sequence and a 403residue cytoplasmic domain containing a tyrosine kinase. The N-terminalhalf of each ectodomain monomer consists of two homologous leucine-richrepeat domains (L1 and L2) of approximately 150 amino acids, separatedby a cysteine-rich region (CR), also approximately 150 amino acids insize. The C-terminal half of each ectodomain monomer (approximately 460residues) consists of three fibronectin type III domains (FnIII-1,FnIII-2 and FnIII-3). The FnIII-2 domain contains an insert domain (ID)of approximately 120 residues, within which lies the furin cleavage sitethat generates the α and β chains of the mature receptor.Intracellularly, each monomer contains a tyrosine kinase catalyticdomain flanked by two regulatory regions (the juxtmembrane region andthe C-tail) that contain the phosphotyrosine binding sites for signalingmolecules (Ward et al, Acta Physiol. 192: 3-9, 2008).

Current models for insulin binding proposes that, in the basal state,the INSR homodimer contains two identical pairs of binding sites(referred to as Site 1 and Site 2) on each monomer (De Meyts, Bioessays,26: 1351-1362, 2004). Binding of insulin to a low affinity site (Site 1)on one α-subunit is followed by a second binding event between the boundinsulin and a different region of the second INSR α-subunit (Site 2).This ligand-mediated bridging between the two a subunits generates thehigh affinity state that results in signal transduction. In contrast,soluble INSR ectodomain, which is not tethered at its C-terminus, cannotgenerate the high affinity receptor-ligand complex. It can bind twomolecules of insulin simultaneously at its two Site 1's, but only withlow affinity (Adams et al, Cell. Mol. Life Sci. 57: 1050-1093, 2000).Site 1 is thought to be comprised of elements from the central β-sheetof the L1 domain and the last 16 residues of the α-chain (referred to asthe CT peptide). Site 2 most likely includes the loops at the junctionof FnIII-1 and FnIII-2. Insulin binding is thought to involve structuralchanges in both insulin and its receptor (Ward and Lawrence, BioEssays31: 422-434, 2009).

Once an insulin molecule has docked onto the receptor and effected itsaction, it may be released back into the extracellular environment or itmay be degraded by the cell. Degradation normally involves endocytosisof the insulin-INSR complex followed by the action of insulin degradingenzyme. Most insulin molecules are degraded by liver cells. It has beenestimated that a typical insulin molecule is finally degraded about 71minutes after its initial release into circulation (Duckworth et al,Endocr. Rev. 19(5): 608-24, 1998).

Insulin Signaling

Insulin induces a signaling network of molecules, carrying theinformation from the INSR to the effector proteins involved inmetabolism and growth. Insulin binding to INSR induces a conformationalchange that promotes activation of an intrinsic tyrosine kinaseactivity, leading to autophosphorylation of the INSR β-subunit. Insulinreceptor substrate (IRS) proteins are recruited to the plasma membranethrough an interaction with the phosphorylated INSR, and these alsobecome phosphorylated on tyrosine residues, promoting recruitment ofadditional signaling proteins to the complex resulting in signalingthrough two major pathways (1) the PI3 kinase/PDK1/PKB pathway whichprimarily regulates metabolism, with some influence on growth ad (2) theRas/ERK mitogenic pathway which primarily regulates cell growth.

Certain marketed insulin analogues have been reported to displayIGF-1-like mitogenic and anti-apoptotic activities in cultured cancercells, raising questions over their long-term safety in humans(Weinstein et al, Diabetes Metab Res Rev 25: 41-49, 2009). Therefore, itwould be desirable to obtain an INSR agonist that did not alter thebalance in metabolic vs. mitogenic INSR signaling, or promoted metabolicsignaling preferentially over mitogenic INSR signaling.

Methods of Identifying Antibodies that are Modulators

The invention provides methods of identifying a candidate polypeptidebinding agent, e.g., an antibody, that modulates binding between firstand second components of a signaling complex, e.g., a receptor such asthe insulin receptor and its ligand insulin.

Without being bound by a theory of the invention, the present disclosureprovides that kinetic perturbation of an interaction between twocomponents (first component, C1 and second component, C2) of a signalingcomplex with a modulator (M) can be described mathematically as:

$K_{C\; 1C\; 2}^{\prime} = {K_{C\; 1C\; 2}\frac{\left( {1 + {M\text{/}K_{{MC}\; 1}}} \right)\left( {1 + {M\text{/}K_{{MC}\; 2}}} \right)}{\left( {1 + {M\text{/}K_{{\lbrack{C\; 1C\; 2}\rbrack}M}}} \right)}}$where the change in binding equilibrium constant between the components(K′_(C1C2)) is a function of equilibrium constant between the components(K_(C1C2)), modulator concentration (M), modulator affinity for thecomplex (K_([C1C2]M)) and modulator affinity for either the firstcomponent (K_(MC1)) or the second component (K_(MC2)).

In cases where the signaling complex is a receptor-ligand complex, andthe modulator is an antibody, the kinetic perturbation of thereceptor-ligand interaction with an antibody can be describedmathematically as:

$K_{RL}^{\prime} = {K_{RL}\frac{\left( {1 + \frac{A}{K_{AR}}} \right)\left( {1 + \frac{A}{K_{AL}}} \right)}{\left( {1 + \frac{A}{K_{RLA}}} \right)}}$where the change in receptor-ligand binding equilibrium constant(K′_(RL)) is a function of receptor-ligand equilibrium constant(K_(RL)), antibody concentration (A), antibody affinity for the complex(K_([RL]A)) and antibody affinity for either the receptor (K_(AR)) orligand (K_(AL)).

A modulator binds the target, or its signaling partner, or a complex ofthe target and signaling partner, in such a manner that the bindingaffinity or binding rate parameter of the target for its signalingpartner is weakened or strengthened. For example, where the target iseither a receptor or ligand, the binding affinity or binding rateparameter of the ligand for its receptor is weakened or strengthened inthe presence of the modulator. A modulator with complete blockingactivity represents a boundary condition in this analysis, since whenK_([C1C2]) is sufficiently high, K′_(C1C2) approaches infinity. Oneimplication of this model is that the degree of signaling modulation isindependent of modulator concentration when the concentration ofmodulator ([M]) is sufficiently above the equilibrium dissociationconstant (K_(D)) for the modulator/antigen interaction to be saturatingfor binding ligand. Hence, modulation of the interaction is related tothe ratio of affinities for the complex versus the components where[M]>K_(D) for the modulator and its antigen.

The present disclosure provides that the biophysical properties of amodulator's interactions with a target and/or its signaling partner canbe used to predict the functional effect of the modulator on the targetsignaling pathway. Modulators which alter the signaling pathway cantherefore be identified based on their relative affinity for target(and/or its signaling partner) in complexed versus uncomplexed form. Theinvention contemplates that kinetic perturbation of an interactionbetween two components (first component, C1 and second component, C2) ofa signaling complex with a modulator (M) can be predicted in thefollowing manner:

-   -   K_([C1C2]M) or K_([MC2]C1) or K_([MC1]C2)<K_(MC2) or K_(MC1)        leads to positive modulation    -   K_([C1C2]M) or K_([MC2]C1) or K_([MC1]C2)=K_(MC2) or K_(MC1)        leads to no modulation    -   K_([C1C2]M) or K_([MC2]C1) or K_([MC1]C2)>K_(MC2) or K_(MC1)        leads to negative modulation

In cases where the signaling complex is a receptor (R)-ligand (L)complex, and the kinetic modulator is an antibody (A), the kineticperturbation can be predicted in the following manner:

-   -   K_([RL]A) or K_([AL]R) or K_([AR]L)<K_(AL) or K_(AR) leads to        positive kinetic modulation    -   K_([RL]A) or K_([AL]R) or K_([AR]L)=K_(AL) or K_(AR) leads to no        kinetic modulation    -   K_([RL]A) or K_([AL]R) or K_([AR]L)>K_(AL) or K_(AR) leads to        negative kinetic modulation

In some embodiments, a modulator, such as an antibody (A) can beidentified by its ability to alter a binding interaction, such as areceptor (R)-ligand (L) interaction at any given sub-saturatingconcentration of the first or second component (e.g. ligand (L)concentration). A modulator antibody or polypeptide could effectivelyshift the affinity and the corresponding dose response of the receptorligand interaction from the 500 pM interaction to either the 10 pM(positive modulator) or 10 nM (negative modulator) as depicted. In someembodiments the modulator will produce a higher level of R-L binding ata given ligand concentration, shifting the assay curve to the left(positive modulation). In other embodiments the modulator will produce alower level of R-L binding at a given ligand concentration, shifting theassay curve to the right (negative modulation). In some embodiments theshift is uniform. In other embodiments the shift is non-uniform,reflecting the involvement of other factors e.g. accessory proteins inthe complex, receptor internalization, etc.

The binding properties of the interaction(s) between the modulator andthe target, its signaling partner and/or a complex comprising the targetand its signaling partner, are generally predictive of the functionaleffect of the kinetic modulator on the target signaling pathway.Depending on the target being studied, certain other factors may need tobe considered. These include: (1) the concentration of the kineticmodulator, the concentration of the target, and/or the concentration ofits signaling partner (e.g., the prediction is optimized if themodulator concentration (ND is significantly greater than the K_(D) ofthe binding between modulator and its antigen), (2) the structural formof the modulator used e.g. monovalent vs. divalent or bivalent, (3)inter/intra target crosslinking, which may restrict the conformation oftarget and/or cause target activation, (4) the modulator's ability toalter assembly or docking, or to alter additional components of thesignaling complex by steric or allosteric mechanisms, (5) signalingpathway specific properties such as alterations in the signal pathwaydue to disease that introduce a “bottleneck,” (6) negative/positivefeedback regulation of the signaling pathway, (7) alteration ofclearance/internalization rates of the components of the signalingcomplex, (8) alterations in the target that uncouple or differentiallyalter ligand binding and activation e.g. a modulator enhances ligandbinding but traps its receptor in a desensitized state, or a modulatorattenuates ligand binding but induces a conformational change in itsreceptor that is activating.

In some aspects the invention provides methods for measuring thedifferential binding of a first component of a signaling complex for asecond component of the signaling complex in the presence or absence ofa test polypeptide agent. In these aspects, differential binding ispreferably observed when there are sub-saturating concentrations of thefirst or second component. In some preferred embodiments theconcentration of the first or second component may be reduced to providesub-saturating conditions.

In some aspects the invention provides methods for measuring thedifferential binding of a test polypeptide binding agent, e.g. antibody,to target and/or its signaling partner, in complexed and uncomplexedform. In these aspects, differential binding is preferably observed whenthere are sub-saturating concentrations of test polypeptide bindingagent. In some preferred embodiments the concentration of testpolypeptide binding agent may be reduced to provide sub-saturatingconditions.

In some embodiments, testing in the absence of a test polypeptide agentis performed using a control compound which is preferably a compoundbelonging to a similar structural class as the test polypeptide agent,but which binds to a different antigen that has no effect on thesignaling complex being tested. For example, a control for a testantibody may be an isotype-matched antibody binding to an unrelatedantigen, e.g. keyhole limpet hemocyanin (KLH).

For positive modulators, at a given sub-saturating concentration of C1,higher C1 affinity will be reflected in a higher signal for C1 bindingto C2 in the presence of the positive modulator. Preferential binding ofthe modulator will be reflected in a higher signal for the complexcomprising C1 and C2, compared to the signal for either C1 alone or C2alone. In some aspects, there may be binding of the modulator to thecomplex of C1 and C2, but no measurable binding to either C1 alone or C2alone.

For negative modulators, at a given sub-saturating concentration of C1,lower C1 affinity will be reflected in a lower signal for C1 binding toC2 in the presence of the modulator. Preferential binding of themodulator will be reflected in a higher signal for binding of themodulator to C1 alone, or to C2 alone, compared to the signal forbinding of the modulator to the complex of C1 and C2.

The invention provides methods of identifying a candidate polypeptidebinding agent, e.g., an antibody, that modulates binding between firstand second components of a signaling complex. In some embodiments, thefirst and second components are polypeptides. In exemplary specificembodiments, the first and second components are endogenous.

In one aspect, the methods of identifying a candidate modulatingantibody include (a) measuring a binding affinity or binding rateparameter of said first component for said second component, in thepresence of a test polypeptide binding agent, e.g. antibody, (b)measuring a binding affinity or binding rate parameter of said firstcomponent for said second component in the absence of said testpolypeptide binding agent; and (c) identifying said test polypeptidebinding agent as a candidate modulating drug when said test polypeptidebinding agent exhibits at least a 1.5-fold difference in the bindingaffinity or binding rate parameter measured in steps (a) and (b). Insome embodiments, the difference in binding affinity or binding rateparameter ranges from about 1.5-fold (i.e., 50%) to about 1000-fold, orabout 1.5-fold to about 100-fold, or about 2-fold to 25-fold, or about2-fold to about 50-fold, or about 1.5-fold to about 25-fold, or about1.5-fold to about 50-fold.

In some embodiments, the test polypeptide binding agent is identified asa candidate positive modulator if the test polypeptide agent strengthensthe binding affinity or binding rate parameter between said firstcomponent and said second component. In other embodiments, the testpolypeptide agent is identified as a candidate negative modulator if thetest polypeptide agent weakens the binding affinity or binding rateparameter between said first component and said second component.

Whether a change (increase or decrease) in a particular binding affinityvalue or binding rate parameter value represents “strengthened” (orstronger) or “weakened” (or weaker) binding affinity or binding rateparameter depends on the value of the parameter and its units, and iswell known in the art. For example, in the case of the parameter K_(A),higher values mean “strengthened” binding affinity, such that a K_(A) ofabout 10⁶M⁻¹ is stronger than a K_(A) of about 10⁵M⁻¹. As anotherexample, in the case of the parameter K_(D), lower values mean“strengthened” binding affinity, such that a K_(D) of about 10⁻⁶M isstronger than a K_(D) of about 10⁻⁵M. Conversely, in the case of K_(A),lower values mean “weakened” binding affinity, such that a K_(A) ofabout 10⁵M⁻¹ is a weakened binding affinity compared to a K_(A) of about10⁶M⁻¹. As another example, in the case of K_(D), higher values mean“weakened” binding affinity, such that a K_(D) of about 10⁻⁵M isweakened binding affinity compared to a K_(D) of about 10⁻⁶M.

As used herein, a “strengthened” binding rate parameter means increasedresidency time, stronger association or weaker dissociation. As usedherein, a “weakened” binding rate parameter means decreased residencytime, weaker association or stronger dissociation.

Binding affinity can also be determined through the ratio of the on-rateand off-rate binding rate parameters. Generally, in the case of on-rate,higher values mean faster or stronger association or increased residencetime, and typically result in stronger binding affinity. Conversely,lower values for on-rate mean slower or weaker association or decreasedresidence time, and typically result in weaker binding affinity.Generally, in the case of off-rate, higher values mean fasterdissociation or decreased residence time, and typically result in weakerbinding affinity. Conversely, lower values for off-rate mean slowerdissociation or increased residence time, and typically result instronger binding affinity. This is because the ratio of off-rate toon-rate, or on-rate to off-rate, indicates binding affinity as displayedin the equations below.

${Affinity}\left\{ {{\begin{matrix}{K_{D} = {\frac{\lbrack A\rbrack\lbrack L\rbrack}{\lbrack{AL}\rbrack} = \frac{{off}\text{-}{rate}}{{on}\text{-}{rate}}}} \\{K_{A} = {\frac{\lbrack{AL}\rbrack}{\lbrack A\rbrack\lbrack L\rbrack} = \frac{{off}\text{-}{rate}}{{on}\text{-}{rate}}}}\end{matrix}{where}A} + {L\begin{matrix}\overset{\mspace{20mu} K_{{o\; n}\mspace{34mu}}}{\rightarrow} \\\underset{K_{off}}{\leftarrow}\end{matrix}{AL}}} \right.$

Even when binding affinity is not detectably or significantly altered,however, the change in residence time, i.e. an increased residence timevia increased on-rate or decreased off-rate, or a decreased residencetime via a decreased on-rate or increased off-rate, may still result indifferential activation of signaling pathways. For example, in someinstances where a receptor may activate two different pathways, thepathways differ in the degree of receptor activation required for a fulleffect. One signaling pathway can be fully activated at low levels ofreceptor activation or residence time, while full activation of thesecond pathway requires higher levels of receptor activation orresidence time.

The predicted correlation of binding characteristics to functionaleffect is depicted in the table below.

Target Binding Characteristics Functional effect R L R-L KD ratios (pAKTassay shift) − − + K_([RL]A) < K_(R), K_(L) Positive modulation − + +K_([AL]R) < K_(L) Positive modulation + − + K_([AR]L) < K_(R) Positivemodulation − + + K_([AL]R) > K_(L) Negative modulation + − + K_([AR]L) >K_(R) Negative modulation

Illustrative examples of data showing that the functional effects ofanti-INSR antibodies correlate with their binding characteristics areshown in the following table.

Functional effect (pAKT assay, fold- decrease in insulin EC₅₀ relativeto Target Binding isotype Characteristics control Ab R L R-L KD ratiosAb) ^(#) Predicted − − + K_([RL]A) < K_(R), K_(L) Positive modulationAb078 Out of Range*  3.4e−10 3.3 Ab085 No Binding   2e−10 8.9Predicted + − + K_([AR]L) < K_(R) Positive modulation Ab001 1.2e−81.16e−10 103.4 9.7 Ab079 9.6e−9 4.96e−10 19.4 6.7 Ab080 1.2e−8  6.8e−1017.6 8.4 Ab083 7.6e−9 3.76e−10 20.2 8.5 Predicted + − + K_([AR]L) =K_(R) Non- Modulators Ab037 1.08e−10   8e−11 1.4 No change Ab0531.48e−10  9.6e−11 1.5 No change Ab062 1.24e−10 1.08e−10 1.1 No change

Thus, the binding properties of the interaction(s) between the modulatorand the target, its signaling partner and/or a complex comprising thetarget and its signaling partner, are generally predictive of thefunctional effect of the modulator polypeptide on the target signalingpathway.

In another aspect, the methods of identifying a candidate modulatingantibody include (a) (i) measuring a binding affinity or binding rateparameter of a test polypeptide binding agent, e.g. antibody, for saidfirst component in the presence of said second component, or (ii)measuring a binding affinity or binding rate parameter of a testpolypeptide binding agent for said second component in the presence ofsaid first component; and (b) (i) measuring a binding affinity orbinding rate parameter of said test polypeptide binding agent for saidfirst component in the absence of said second component, or (ii)measuring a binding affinity or binding rate parameter of said testpolypeptide binding agent for said second component in the absence ofsaid first component; and (c) identifying said test polypeptide bindingagent as a candidate kinetic modulating drug when said test polypeptidebinding agent exhibits at least a 1.5-fold (i.e., 50%) difference in thebinding affinity or binding rate parameters measured in steps (a) and(b).

In some embodiments, the test polypeptide binding agent is identified asa candidate positive modulator if the binding affinity or binding rateparameter measured in step (a) is at least 1.5-fold (i.e., 50%) strongerthan the binding affinity or binding rate parameter measured in step(b). In specific embodiments, the binding affinity or binding rateparameter measured in step (a) compared to that measured in step (b) isabout 1.5-fold (i.e., 50%) to about 1000-fold stronger for step (a) vs.step (b), or about 1.5-fold to about 100-fold, or about 2-fold to25-fold, or about 2-fold to about 50-fold, or about 1.5-fold to about25-fold, or about 1.5-fold to about 50-fold, e.g., at least 1.5-fold,2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold,19-fold or 20-fold, or up to 500-fold, or up to 200-fold, or up to150-fold, or up to 100-fold, or up to 90-fold, or up to 80-fold, or upto 70-fold, or up to 60-fold, or up to 50-fold, or up to 40-fold, up to30-fold, up to 20-fold, or up to 10-fold.

In other embodiments, the test polypeptide binding agent is identifiedas a candidate negative modulator if the binding affinity or bindingrate parameter measured in step (b) is at least 1.5-fold (i.e., 50%)stronger than the binding affinity or binding rate parameter measured instep (a). In specific embodiments, the binding affinity or binding rateparameter measured in step (b) compared to that measured in step (a) isabout 1.5-fold (i.e., 50%) to about 1000-fold stronger for step (b) vs.step (a), or about 1.5-fold to about 100-fold, or about 2-fold to25-fold, or about 2-fold to about 50-fold, or about 1.5-fold to about25-fold, or about 1.5-fold to about 50-fold, e.g. at least 1.5-fold,2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold,19-fold or 20-fold, or up to 500-fold, or up to 200-fold, or up to150-fold, or up to 100-fold, or up to 90-fold, or up to 80-fold, or upto 70-fold, or up to 60-fold, or up to 50-fold, or up to 40-fold, up to30-fold, up to 20-fold, or up to 10-fold.

In some embodiments, the binding affinity or binding rate parameter ofthe test polypeptide binding agent for the first component alone ismeasured. In some embodiments, the binding affinity or binding rateparameter of the test polypeptide binding agent for the second componentalone is measured.

In some embodiments, the test polypeptide binding agent is identified asa candidate positive modulator if one or more binding affinity orbinding rate parameters selected from the group consisting of (A) thebinding affinity or binding rate parameter of the test polypeptidebinding agent for a complex comprising the first and second components,optionally K_([C1C2]M), (B) the binding affinity or binding rateparameter of the first component for a complex comprising thepolypeptide binding agent and the second component, optionallyK_([MC2]C1,) or (C) the binding affinity or binding rate parameter ofthe second component for a complex comprising the polypeptide bindingagent and the first component, optionally K_([MC1]C2), is at least about1.5-fold stronger than one or more binding affinity or binding rateparameter selected from the group consisting of (1) the binding affinityor binding rate parameter of the test polypeptide binding agent for thesecond component alone, optionally K_(MC2) or (2) the binding affinityor binding rate parameter of the test polypeptide binding agent for thefirst component alone, optionally K_(MC1). In some embodiments, thespecific binding affinity or binding rate parameter of any one or moreof (A), (B) or (C) is about 1.5-fold (i.e., 50%) to about 1000-foldstronger than the binding affinity or binding rate parameter of any oneor more of (1) or (2); or alternatively, about 1.5-fold to about100-fold stronger, or about 2-fold to 25-fold, or about 2-fold to about50-fold, or about 1.5-fold to about 25-fold, or about 1.5-fold to about50-fold, e.g. at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold,7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold,15-fold, 16-fold, 17-fold, 18-fold, 19-fold or 20-fold, or up to500-fold, or up to 200-fold, or up to 150-fold, or up to 100-fold, or upto 90-fold, or up to 80-fold, or up to 70-fold, or up to 60-fold, or upto 50-fold, or up to 40-fold, up to 30-fold, up to 20-fold, or up to10-fold. For example, in some embodiments, the binding affinity orbinding rate parameter of any one or more of (A), (B) or (C) is strongerthan the binding affinity or binding rate parameter of both (1) and (2).In some embodiments, the binding affinity or binding rate parameter of(1) is stronger than the binding affinity or binding rate parameter of(2). In other embodiments, the binding affinity or binding rateparameter of (2) is stronger than the binding affinity or binding rateparameter of (1). In some embodiments, two or more binding affinity orbinding rate parameters are measured and compared, e.g. off-rate andon-rate, or K_(A) and K_(D), or any combination thereof.

In specific embodiments, wherein the binding affinity measured is theequilibrium dissociation constant K_(D), any of K_([C1C2]M),K_([MC2]C1), or K_([MC1]C2) is lower, e.g., about 1.5-fold to 1000-foldlower, than any of K_(MC2) or K_(MC1). Similarly, wherein the bindingaffinity measured is the off-rate, any of the off-rates between (A)[C1C2] and M, or (B) [MC2] and C1, or (C) [MC1] and C2 are lower, e.g.about 1.5-fold to 1000-fold lower, than any of the off-rates between (1)M and C2 or (2) M and C1. In one exemplary embodiment, K_([C1C2]m) isabout 1.5-fold to 1000-fold lower than K_(MC2). In another exemplaryembodiment, K_([MC2]C1) is about 1.5-fold to 1000-fold lower thanK_(MC2). In another exemplary embodiment, K_([MC1]C2) is about 1.5-foldto 1000-fold lower than K_(MC2). In another exemplary embodiment,K_([C1C2]m) is about 1.5-fold to 1000-fold lower than K_(MC1). Inanother exemplary embodiment, K_([MC2]C1) is about 1.5-fold to 1000-foldlower than K_(MC1). In yet another exemplary embodiment, K_([MC1]C2) isabout 1.5-fold to 1000-fold lower than K_(MC1). Similar examples can beenvisioned for each of the off-rates between (A) [C1C2] and M, or (B)[MC2] and C1, or (C) [MC1] and C2, compared to each of the off-ratesbetween (1) M and C2 or (2) M and C1.

Conversely, where the binding affinity measured is the equilibriumassociation constant K_(A), any of K_([C1C2]M), K_([MC2]C1), orK_([MC1]C2) is higher, e.g., about 1.5-fold to 1000-fold higher, thanany of K_(MC2) or K_(MC1). Similarly, wherein the binding affinitymeasured is the on-rate, any of the on-rates between (A) [C1C2] and M,or (B) [MC2] and C1, or (C) [MC1] and C2 are higher, e.g. about 1.5-foldto 1000-fold higher, than any of the on-rates between (1) M and C2 or(2) M and C1. In one exemplary embodiment, K_([C1C2]m) is about 1.5-foldto 1000-fold higher than K_(MC2). In another exemplary embodiment,K_([MC2]C1) is about 1.5-fold to 1000-fold higher than K_(MC2). Inanother exemplary embodiment, K_([MC1]C2) is about 1.5-fold to 1000-foldhigher than K_(MC2). In another exemplary embodiment, K_([C1C2]M) isabout 1.5-fold to 1000-fold higher than K_(MC1). In another exemplaryembodiment, K_([MC2]C1) is about 1.5-fold to 1000-fold higher thanK_(MC1). In yet another exemplary embodiment, K_([MC1]C2) is about1.5-fold to 1000-fold higher than K_(MC1). Similar examples can beenvisioned for each of the on-rates between (A) [C1C2] and M, or (B)[MC2] and C1, or (C) [MC1] and C2, compared to each of the on-ratesbetween (1) M and C2 or (2) M and C1.

In some embodiments, the test polypeptide binding agent is identified asa candidate negative modulator if one or more binding affinity orbinding rate parameters selected from the group consisting of (1) thebinding affinity or binding rate parameter of the test polypeptidebinding agent for the second component alone, optionally K_(MC2), or (2)the binding affinity or binding rate parameter of the test polypeptidebinding agent for the first component alone, optionally K_(MC1), is atleast about 1.5-fold stronger than one or more binding affinity orbinding rate parameter selected from the group consisting of (A) thebinding affinity or binding rate parameter of the test polypeptidebinding agent for a complex comprising the first and second components,optionally K_([C1C2]M), (B) the binding affinity or binding rateparameter of the first component for a complex comprising thepolypeptide binding agent and the second component, optionallyK_([MC2]C1,) or (C) the binding affinity or binding rate parameter ofthe second component for a complex comprising the polypeptide bindingagent and the first component, optionally K_([MC1]C2). In someembodiments, the specific binding affinity or binding rate parameter ofany one or more of (1) or (2) is about 1.5-fold (i.e., 50%) to about1000-fold stronger than the binding affinity or binding rate parameterof any one or more of (A), (B) or (C); or alternatively, about 1.5-foldto about 100-fold stronger, or about 2-fold to 25-fold, or about 2-foldto about 50-fold, or about 1.5-fold to about 25-fold, or about 1.5-foldto about 50-fold, e.g., at least 1.5-fold, 2-fold, 3-fold, 4-fold,5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold,13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold or20-fold, or up to 500-fold, or up to 200-fold, or up to 150-fold, or upto 100-fold, or up to 90-fold, or up to 80-fold, or up to 70-fold, or upto 60-fold, or up to 50-fold, or up to 40-fold, up to 30-fold, up to20-fold, or up to 10-fold. In some embodiments, the binding affinity orbinding rate parameter of any of (1) or (2) is stronger than the bindingaffinity or binding rate parameter of all of (A), (B) and (C). In someembodiments, the binding affinity or binding rate parameter of (1) isstronger than the binding affinity or binding rate parameter of (2). Inother embodiments, the binding affinity or binding rate parameter of (2)is stronger than the binding affinity or binding rate parameter of (1).In some embodiments, two or more binding affinity or binding rateparameters are measured and compared, e.g. off-rate and on-rate, orK_(A) and K_(D), or any combination thereof.

In specific embodiments, where the binding affinity measured is theequilibrium dissociation constant K_(D), any of K_(MC2) or K_(MC1) islower, e.g., about 1.5-fold to 1000-fold lower, than any of K_([C1C2]M),K_([MC2]C1), or K_([MC1]C2). Similarly, wherein the binding affinitymeasured is the off-rate, any of the off-rates between (1) M and C2 or(2) M and C1 are lower, e.g. about 1.5-fold to 1000-fold lower, than anyof the off-rates between (A) [C1C2] and M, or (B) [MC2] and C1, or (C)[MC1] and C2. In one exemplary embodiment K_(MC2) is about 1.5-fold to1000-fold lower than K_([C1C2]C1). In another exemplary embodiment,K_(MC2) is about 1.5-fold to 1000-fold lower than K_([MC2]C1). Inanother exemplary embodiment, K_(MC2) is about 1.5-fold to 1000-foldlower than K_([MC1]C2). In another exemplary embodiment, K_(MC1) isabout 1.5-fold to 1000-fold lower than K_([C1C2]M). In another exemplaryembodiment, K_(MC1) is about 1.5-fold to 1000-fold lower thanK_([MC2]C1). In yet another exemplary embodiment, K_(MC1) is about1.5-fold to 1000-fold lower than K_([MC1]C2). Similar examples can beenvisioned for each of the off-rates between (1) M and C2 or (2) M andC1, compared to each of the off-rates between (A) [C1C2] and M, or (B)[MC2] and C1, or (C) [MC1] and C2.

Conversely, wherein the binding affinity is the equilibrium associationconstant K_(A), any of K_(MC2) or K_(MC1) is higher, e.g., about1.5-fold to 1000-fold higher, than any of K_([C1C2]M), K_([MC2]C1), orK_([MC1]C)2. Similarly, wherein the binding affinity measured is theon-rate, any of the on-rates between (1) M and C2 or (2) M and C1 arehigher, e.g. about 1.5-fold to 1000-fold higher, than any of theon-rates between (A) [C1C2] and M, or (B) [MC2] and C1, or (C) [MC1] andC2. In one exemplary embodiment K_(MC2) is about 1.5-fold to 1000-foldhigher than K_([C1C2]M). In another exemplary embodiment, K_(MC2) isabout 1.5-fold to 1000-fold higher than K_([MC2]C1). In anotherexemplary embodiment, K_(MC2) is about 1.5-fold to 1000-fold higher thanK_([MC1]C2). In another exemplary embodiment, K_(MC1) is about 1.5-foldto 1000-fold higher than K_([C1C2]M). In another exemplary embodiment,K_(MC1) is about 1.5-fold to 1000-fold higher than K_([MC2]C1). In yetanother exemplary embodiment, K_(MC1) is about 1.5-fold to 1000-foldhigher than K_([MC1]C2). Similar examples can be envisioned for each ofthe on-rates between (1) M and C2 or (2) M and C1, compared to each ofthe on-rates between (A) [C1C2] and M, or (B) [MC2] and C1, or (C) [MC1]and C2.

In certain embodiments, the modulator is an antibody and C1 and C2 areselected from the group consisting of insulin and insulin receptor.

In any of these embodiments, the test polypeptide binding agent andsecond component can be contacted with multiple different concentrationsof said first component. In any of these embodiments, the testpolypeptide binding agent and first component can be contacted withmultiple different concentrations of said second component. In any ofthese embodiments, multiple different concentrations of the testpolypeptide binding agent can be contacted with said first component andsaid second component.

When the effect of test polypeptide binding agent on the bindinginteraction between the first component and second component isdetermined, in some specific embodiments, when the antigen for the testpolypeptide binding agent is the first component, e.g., ligand, the testpolypeptide binding agent is at a saturating concentration compared tothe concentration of the first component. Alternatively, when theantigen for the test polypeptide binding agent is the second component,e.g., receptor, the test polypeptide binding agent is at a saturatingconcentration compared to the concentration of the second component. Insome embodiments, the concentration of the test polypeptide bindingagent is greater than or equal to the K_(D) of the test polypeptidebinding agent for a complex comprising the first component and thesecond component. In further embodiments, the concentration of thesecond component is less than the K_(D) of the test polypeptide bindingagent for the first component, e.g., ligand. In yet further embodiments,the concentration of the first component, e.g., ligand, is at asubsaturating concentration for the binding of first component to secondcomponent, e.g., receptor. In some embodiments, the concentration of thefirst component, e.g., ligand is within the range of about EC₂₀ to EC₈₀for the interaction of the first component with the second component. Insome embodiments, one or more concentrations of the test polypeptidebinding agent is contacted with multiple different concentrations of thefirst component, e.g., ligand, in the presence of one or moreconcentrations of the second component, e.g., receptor. In someembodiments, one or more concentrations of the test polypeptide bindingagent is contacted with multiple different concentrations of the secondcomponent, e.g., receptor, in the presence of one or more concentrationsof the first component, e.g., ligand.

When differential binding of test polypeptide binding agent to complexedvs. uncomplexed target and/or signaling partner is determined in orderto identify a positive modulator, in some embodiments, the testpolypeptide binding agent is at a saturating concentration for a complexcomprising the first component and the second component. In someembodiments, the concentration of test polypeptide binding agent isgreater than or equal to the K_(D) of the test polypeptide binding agentfor a complex comprising the first component, e.g., ligand, and thesecond component, e.g., receptor. In further embodiments, theconcentration of the second component, e.g., receptor is greater thanthe K_(D) of the second component, e.g., receptor, for the firstcomponent, e.g., ligand. In further embodiments, the concentration ofthe first component, e.g., ligand, is a saturating concentration for thesecond component, e.g., receptor. In yet further embodiments, the testpolypeptide binding agent is at a subsaturating concentration for acomplex comprising the first component and the second component. In someembodiments, the concentration of the polypeptide binding agent iswithin the range of about EC₂₀ to EC₈₀ for the interaction of the firstcomponent with the second component. In some embodiments, theconcentration of the second component, e.g., receptor, is greater thanthe K_(D) of the second component, e.g., receptor, for the firstcomponent, e.g., ligand. In some embodiments, the concentration of thefirst component, e.g., ligand, is a saturating concentration for thesecond component, e.g., receptor.

When differential binding of test polypeptide binding agent to complexedvs. uncomplexed target and/or signaling partner is determined in orderto identify a negative modulator, in some embodiments, when the antigento which the test polypeptide binding agent binds is the firstcomponent, e.g., ligand, the test polypeptide binding agent is at asubsaturating concentration for the first component. When the antigen towhich the test polypeptide binding agent binds is the second component,e.g., receptor, the test polypeptide binding agent is at a subsaturatingconcentration for the second component. In further embodiments, theconcentration of the polypeptide binding agent is within the range ofabout EC₂₀ to EC₈₀ for the interaction of the first component with thesecond component. In further embodiments, the concentration of thesecond component, e.g., receptor, is greater than the K_(D) of thesecond component, e.g., receptor, for the first component, e.g., ligand.In further embodiments, the concentration of the first component, e.g.,ligand, is a saturating concentration for the second component, e.g.,receptor.

In some embodiments, the methods further involve assaying a plurality oftest polypeptide binding agents, e.g. antibodies, for binding affinityto any one of (a) the first component, (b) the second component, or (c)a complex comprising the first component and second component. In somespecific embodiments, the polypeptide binding agents have a bindingaffinity characterized, e.g., by an equilibrium dissociation constantK_(D) of about 10⁻⁵M or less, or about 10⁻⁶M or less, or about 10⁻⁷M orless, or about 10⁻⁸M or less, where a lower K_(D) means stronger bindingaffinity. In some embodiments, the plurality of test polypeptide bindingagents screened are variants of a parent polypeptide binding agent madeby introducing one or more different mutations into a parent polypeptidebinding agent.

In further embodiments, the polypeptide binding agents may be screenedfor selectivity of effect for the first or second component, compared toa different binding partner such as a decoy receptor, clearancereceptor, or alternate signal pathway component. Such methods mayinvolve identifying a polypeptide binding agent that does notsignificantly change the binding affinity or binding rate parameter ofthe first or second component for a different binding partner, suchbinding partner being neither the first nor second component. In someembodiments, the presence of the polypeptide binding agent changes thebinding affinity or binding rate parameter of the first or secondcomponent for a different binding partner no more than 5-fold, or nomore than 10-fold, or no more than 20-fold, or no more than 30-fold, orno more than 40-fold, or no more than 50-fold.

Any of the preceding methods may further include measuring the level ofsignaling mediated by the signaling complex in the presence and absenceof the test polypeptide binding agent, and determining whether the testpolypeptide binding agent is additionally an agonist, partial agonist,antagonist or partial antagonist. Antagonism or agonism can be measuredin any in vitro or in vivo assay known in the art, including but notlimited to signaling in a phosphorylation assay, ion flux assay,molecular transport assay, or gene expression assay.

In some embodiments, the test polypeptide binding agent shifts(positively or negatively) the dose-response curve of the interaction ofthe first component, e.g. ligand, with the second component, e.g.receptor. The shift may manifest as an increased or decreased EC₅₀ by atleast about 1.5-fold, e.g. about 1.5-fold to about 100-fold. In someembodiments, the test polypeptide binding agent does not significantlychange the maximal agonist response of the signal produced byinteraction of the first and second components of the signaling complex.In other embodiments, the test polypeptide binding agent itself acts asan antagonist (e.g., reduces the maximal agonist response of thesignaling produced by said signaling complex) or agonist (e.g. increasesthe maximal agonist response of the signaling produced by said signalingcomplex).

Where the test polypeptide binding agent acts as an antagonist orpartial antagonist, the maximal agonist response may be decreased, e.g.,by about 1.5-fold to about 100-fold, or about 2-fold to about 25-fold,or about 1.5-fold to about 50-fold; or, decreased by about 10%, 25%, 50%(1.5-fold), 75%, 2-fold, 3-fold, or 4-, 5-, 6-, 7-, 8-, 9- or 10-fold.Alternatively, where the test polypeptide binding agent acts as anagonist or partial agonist, the maximal agonist response may beincreased, e.g. by at least about 10%, 25%, 50% (1.5-fold), 75%, 2-fold,3-fold, or 4-, 5-, 6-, 7-, 8-, 9- or 10-fold. Moreover, when the testpolypeptide binding agent acts as an antagonist or partial antagonist,the IC50 may be 1×10⁻⁵ or less. The test polypeptide binding agent mayexhibit further desirable characteristics, e.g. the test polypeptidebinding agent does not significantly decrease clearance of said firstcomponent, or said second component, or said signaling complexcomprising said first and second components.

Methods of identifying modulating agents, e.g. kinetic modulatingagents, are described further in co-pending, co-owned U.S. PatentApplication No. 61/246,079, filed Sep. 25, 2009, U.S. Patent ApplicationNo. 62/306,324, filed Feb. 19, 2010, and International PatentApplication No. PCT/US10/50312 filed Sep. 24, 2010.

The test polypeptide binding agent may exhibit further desirablecharacteristics, e.g. the test polypeptide binding agent does notsignificantly decrease clearance of said first component, or said secondcomponent, or said signaling complex comprising said first and secondcomponents.

In a related aspect, the invention provides methods of identifyingmodulators of the insulin/insulin receptor signaling complex and anantibody or other modulator identified by any of the methods describedabove or anywhere in the present application.

Types and Sources of Antibodies

The present invention encompasses target specific antibodies that bindto insulin, insulin receptor or the insulin/insulin receptor complex. Inexemplary embodiments, a target specific antibody of the invention cancomprise a human kappa (κ) or a human lambda (λ) light chain or an aminoacid sequence derived therefrom, or a human heavy chain or a sequencederived therefrom, or both heavy and light chains together in a singlechain, dimeric, tetrameric or other form. In some embodiments, a heavychain and a light chain of a target specific immunoglobulin aredifferent amino acid molecules. In other embodiments, the same aminoacid molecule contains a heavy chain variable region and a light chainvariable region of a target specific antibody.

The term “antibody” is used in the broadest sense and includes fullyassembled antibodies, tetrameric antibodies, monoclonal antibodies,polyclonal antibodies, multispecific antibodies (e.g., bispecificantibodies), human and humanized antibodies, antibody fragments that canbind an antigen (e.g., Fab′, F′(ab)2, Fv, single chain antibodies,diabodies), and recombinant peptides comprising the forgoing as long asthey exhibit the desired biological activity. An “immunoglobulin” or“tetrameric antibody” is a tetrameric glycoprotein that consists of twoheavy chains and two light chains, each comprising a variable region anda constant region. Antigen-binding portions may be produced byrecombinant DNA techniques or by enzymatic or chemical cleavage ofintact antibodies. Antibody fragments or antigen-binding portionsinclude, inter alia, Fab, Fab′, F(ab′)2, Fv, domain antibody (dAb),complementarity determining region (CDR) fragments, single-chainantibodies (scFv), single chain antibody fragments, chimeric antibodies,diabodies, triabodies, tetrabodies, minibody, linear antibody; chelatingrecombinant antibody, a tribody or bibody, an intrabody, a nanobody, asmall modular immunopharmaceutical (SMIP), a antigen-binding-domainimmunoglobulin fusion protein, a camelized antibody, a VHH containingantibody, or a variant or a derivative thereof, and polypeptides thatcontain at least a portion of an immunoglobulin that is sufficient toconfer specific antigen binding to the polypeptide, as long as theantibody retains the desired biological activity.

In a naturally-occurring immunoglobulin, each tetramer is composed oftwo identical pairs of polypeptide chains, each pair having one “light”(about 25 kDa) and one “heavy” chain (about 50-70 kDa). Theamino-terminal portion of each chain includes a variable region of about100 to 110 or more amino acids primarily responsible for antigenrecognition. The carboxy-terminal portion of each chain defines aconstant region primarily responsible for effector function. Human lightchains are classified as kappa (κ) and lambda (λ) light chains. Heavychains are classified as mu (μ), delta (Δ), gamma (γ), alpha (α), andepsilon (ε), and define the antibody's isotype as IgM, IgD, IgG, IgA,and IgE, respectively. Within light and heavy chains, the variable andconstant regions are joined by a “J” region of about 12 or more aminoacids, with the heavy chain also including a “D” region of about 10 moreamino acids. See generally, Fundamental Immunology, Ch. 7 (Paul, W.,ed., 2nd ed. Raven Press, N.Y. (1989)) (incorporated by reference in itsentirety for all purposes). The variable regions of each light/heavychain pair form the antibody binding site such that an intactimmunoglobulin has two binding sites.

Each heavy chain has at one end a variable domain (V_(H)) followed by anumber of constant domains. Each light chain has a variable domain atone end (V_(L)) and a constant domain at its other end; the constantdomain of the light chain is aligned with the first constant domain ofthe heavy chain, and the light chain variable domain is aligned with thevariable domain of the heavy chain. Particular amino acid residues arebelieved to form an interface between the light and heavy chain variabledomains (Chothia et al., J. Mol. Biol. 196:901-917, 1987).

Immunoglobulin variable domains exhibit the same general structure ofrelatively conserved framework regions (FR) joined by threehypervariable regions or CDRs. From N-terminus to C-terminus, both lightand heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3and FR4. The assignment of amino acids to each domain is in accordancewith the definitions of Kabat Sequences of Proteins of ImmunologicalInterest (National Institutes of Health, Bethesda, Md. (1987 and 1991)),or Chothia & Lesk, (J. Mol. Biol. 196:901-917, 1987); Chothia et al.,(Nature 342:878-883, 1989).

The hypervariable region of an antibody refers to the CDR amino acidresidues of an antibody which are responsible for antigen-binding. Thehypervariable region comprises amino acid residues from a CDR [i.e.,residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chainvariable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavychain variable domain as described by Kabat et al., Sequences ofProteins of Immunological Interest, 5^(th) Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)] or those residuesfrom a hypervariable loop (i.e., residues 26-32 (L1), 50-52 (L2) and91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2)and 96-101 (H3) in the heavy chain variable domain as described by[Chothia et al., J. Mol. Biol. 196: 901-917 (1987)]. However, one ofskill in the art understands that the actual location of the CDRresidues may vary from the projected residues described above when thesequence of the particular antibody is identified.

Framework or FR residues are those variable domain residues other thanthe hypervariable region residues.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes, IgA,IgD, IgE, IgG and IgM, which may be further divided into subclasses orisotypes, e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known. Different isotypes have differenteffector functions; for example, IgG1 and IgG3 isotypes have ADCCactivity. An antibody of the invention, if it comprises a constantdomain, may be of any of these subclasses or isotypes.

In exemplary embodiments, an antibody of the invention can comprise ahuman kappa (κ) or a human lambda (λ) light chain or an amino acidsequence derived therefrom, or a human heavy chain or a sequence derivedtherefrom, or both heavy and light chains together in a single chain,dimeric, tetrameric or other form.

Monoclonal antibody refers to an antibody obtained from a population ofsubstantially homogeneous antibodies. Monoclonal antibodies aregenerally highly specific, and may be directed against a singleantigenic site, in contrast to conventional (polyclonal) antibodypreparations that typically include different antibodies directedagainst different determinants (epitopes). In addition to theirspecificity, monoclonal antibodies are advantageous in that they aresynthesized by the homogeneous culture, uncontaminated by otherimmunoglobulins with different specificities and characteristics.

Monoclonal antibodies to be used in accordance with the presentinvention may be made by the hybridoma method first described by Kohleret al., (Nature, 256:495-7, 1975), or may be made by recombinant DNAmethods (see, e.g., U.S. Pat. No. 4,816,567). The monoclonal antibodiesmay also be isolated from phage antibody libraries using the techniquesdescribed in, for example, Clackson et al., (Nature 352:624-628, 1991)and Marks et al., (J. Mol. Biol. 222:581-597, 1991).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster or macaque monkey, is immunized to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the protein used for immunization (Harlow & Lane; Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press: Cold SpringHarbor, N.Y. (1988).

Recombinant Production of Antibodies

The present invention also encompasses nucleic acid molecules encodingantibodies of the invention. In some embodiments, different nucleic acidmolecules encode a heavy chain variable region and a light chainvariable region of an antigen-specific antibody. In other embodiments,the same nucleic acid molecule encodes a heavy chain and a light chainvariable regions of an antigen-specific antibody.

DNA encoding a monoclonal antibody of the invention may be isolated andsequenced from a hybridoma cell secreting the antibody usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the monoclonal antibodies). Sequence determination willgenerally require isolation of at least a portion of the gene or cDNA ofinterest. Usually this requires cloning the DNA or, preferably, mRNA(i.e., cDNA) encoding the monoclonal antibodies. Cloning is carried outusing standard techniques (see, e.g., Sambrook et al. (1989) MolecularCloning: A Laboratory Guide, Vols 1-3, Cold Spring Harbor Press, whichis incorporated herein by reference). For example, a cDNA library may beconstructed by reverse transcription of polyA+ mRNA, preferablymembrane-associated mRNA, and the library screened using probes specificfor human immunoglobulin polypeptide gene sequences. Nucleotide probereactions and other nucleotide hybridization reactions are carried outat conditions enabling the identification of polynucleotides whichhybridize to each other under specified conditions. The hybridizationconditions can be calculated as described in Sambrook, et al., (Eds.),Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress: Cold Spring Harbor, N.Y. (1989), pp. 9.47 to 9.51

In a preferred embodiment, the polymerase chain reaction (PCR) is usedto amplify cDNAs (or portions of full-length cDNAs) encoding animmunoglobulin gene segment of interest (e.g., a light chain variablesegment). The amplified sequences can be readily cloned into anysuitable vector, e.g., expression vectors, minigene vectors, or phagedisplay vectors. It will be appreciated that the particular method ofcloning used is not critical, so long as it is possible to determine thesequence of some portion of the immunoglobulin polypeptide of interest.As used herein, an “isolated” nucleic acid molecule or “isolated”nucleic acid sequence is a nucleic acid molecule that is either (1)identified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe nucleic acid or (2) cloned, amplified, tagged, or otherwisedistinguished from background nucleic acids such that the sequence ofthe nucleic acid of interest can be determined, is considered isolated.An isolated nucleic acid molecule is other than in the form or settingin which it is found in nature. Isolated nucleic acid moleculestherefore are distinguished from the nucleic acid molecule as it existsin natural cells. However, an isolated nucleic acid molecule includes anucleic acid molecule contained in cells that ordinarily express theantibody where, for example, the nucleic acid molecule is in achromosomal location different from that of natural cells.

One source for RNA used for cloning and sequencing is a hybridomaproduced by obtaining a B cell from the transgenic mouse and fusing theB cell to an immortal cell. Alternatively, RNA can be isolated from Bcells (or whole spleen) of the immunized animal. When sources other thanhybridomas are used, it may be desirable to screen for sequencesencoding immunoglobulins or immunoglobulin polypeptides with specificbinding characteristics. One method for such screening is the use ofphage display technology. Phage display is described further herein andis also well-known in the art. See e.g., Dower et al., WO 91/17271,McCafferty et al., WO 92/01047, and Caton and Koprowski, (Proc. Natl.Acad. Sci. USA, 87:6450-54 (1990)), each of which is incorporated hereinby reference. In one embodiment, cDNA from an immunized transgenic mouse(e.g., total spleen cDNA) is isolated, the polymerase chain reaction isused to amplify a cDNA sequences that encode a portion of animmunoglobulin polypeptide, e.g., CDR regions, and the amplifiedsequences are inserted into a phage vector. cDNAs encoding peptides ofinterest, e.g., variable region peptides with desired bindingcharacteristics, are identified by standard phage display techniquessuch as panning.

The sequence of the amplified or cloned nucleic acid is then determined.Typically the sequence encoding an entire variable region of theimmunoglobulin polypeptide is determined, however, it will sometimes byadequate to sequence only a portion of a variable region, for example,the CDR-encoding portion. Typically the portion sequenced will be atleast 30 bases in length, more often based coding for at least aboutone-third or at least about one-half of the length of the variableregion will be sequenced.

Sequencing can be carried out on clones isolated from a cDNA library,or, when PCR is used, after subcloning the amplified sequence or bydirect PCR sequencing of the amplified segment. Sequencing is carriedout using standard techniques (see, e.g., Sambrook et al. (1989)Molecular Cloning: A Laboratory Guide, Vols 1-3, Cold Spring HarborPress, and Sanger, F. et al. (1977) Proc. Natl. Acad. Sci. USA 74:5463-5467, which is incorporated herein by reference). By comparing thesequence of the cloned nucleic acid with published sequences of humanimmunoglobulin genes and cDNAs, one of skill will readily be able todetermine, depending on the region sequenced, (i) the germline segmentusage of the hybridoma immunoglobulin polypeptide (including the isotypeof the heavy chain) and (ii) the sequence of the heavy and light chainvariable regions, including sequences resulting from N-region additionand the process of somatic mutation. One source of immunoglobulin genesequence information is the National Center for BiotechnologyInformation, National Library of Medicine, National Institutes ofHealth, Bethesda, Md.

Once isolated, the DNA may be placed into expression vectors, which arethen transfected into host cells such as E. coli cells, simian COScells, human embryonic kidney 293 cells (e.g., 293E cells), Chinesehamster ovary (CHO) cells, or myeloma cells that do not otherwiseproduce immunoglobulin protein, to obtain the synthesis of monoclonalantibodies in the recombinant host cells. Recombinant production ofantibodies is well known in the art.

Expression control sequences refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

In an alternative embodiment, the amino acid sequence of animmunoglobulin of interest may be determined by direct proteinsequencing. Suitable encoding nucleotide sequences can be designedaccording to a universal codon table.

Amino acid sequence variants of the desired antibody may be prepared byintroducing appropriate nucleotide changes into the encoding DNA, or bypeptide synthesis. Such variants include, for example, deletions from,and/or insertions into and/or substitutions of, residues within theamino acid sequences of the antibodies. Any combination of deletion,insertion, and substitution is made to arrive at the final construct,provided that the final construct possesses the desired characteristics.The amino acid changes also may alter post-translational processes ofthe antibody, such as changing the number or position of glycosylationsites.

Nucleic acid molecules encoding amino acid sequence variants of theantibody are prepared by a variety of methods known in the art. Thesemethods include, but are not limited to, isolation from a natural source(in the case of naturally occurring amino acid sequence variants) orpreparation by oligonucleotide-mediated (or site-directed) mutagenesis,PCR mutagenesis, and cassette mutagenesis of an earlier prepared variantor a non-variant version of the antibody.

The invention also provides isolated nucleic acid encoding antibodies ofthe invention, optionally operably linked to control sequencesrecognized by a host cell, vectors and host cells comprising the nucleicacids, and recombinant techniques for the production of the antibodies,which may comprise culturing the host cell so that the nucleic acid isexpressed and, optionally, recovering the antibody from the host cellculture or culture medium Various systems and methods for antibodyproduction are reviewed by Birch & Racher (Adv. Drug Deliv. Rev. 671-685(2006)).

For recombinant production of the antibody, the nucleic acid encoding itis isolated and inserted into a replicable vector for further cloning(amplification of the DNA) or for expression. DNA encoding themonoclonal antibody is readily isolated and sequenced using conventionalprocedures (e.g., by using oligonucleotide probes that are capable ofbinding specifically to genes encoding the heavy and light chains of theantibody). Many vectors are available. The vector components generallyinclude, but are not limited to, one or more of the following: a signalsequence, an origin of replication, one or more selective marker genes,an enhancer element, a promoter, and a transcription terminationsequence.

Suitable host cells for cloning or expressing the DNA in the vectorsherein are prokaryote, yeast, or higher eukaryote cells. Suitableprokaryotes for this purpose include eubacteria, such as Gram-negativeor Gram-positive organisms, for example, Enterobacteriaceae such asEscherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus,Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratiamarcescans, and Shigella, as well as Bacilli such as B. subtilis and B.licheniformis (e.g., B. licheniformis 41 P disclosed in DD 266,710published Apr. 12, 1989), Pseudomonas such as P. aeruginosa, andStreptomyces. One preferred E. coli cloning host is E. coli 294 (ATCC31,446), although other strains such as E. coli B, E. coli X1776 (ATCC31,537), and E. coli W3110 (ATCC 27,325) are suitable. These examplesare illustrative rather than limiting.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors. Saccharomyces cerevisiae, or common baker'syeast, is the most commonly used among lower eukaryotic hostmicroorganisms. However, a number of other genera, species, and strainsare commonly available and useful herein, such as Schizosaccharomycespombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K.waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans,and K. marxianus; yarrowia (EP 402,226); Pichia pastors (EP 183,070);Candida; Trichoderma reesia (EP 244,234); Neurospora crassa;Schwanniomyces such as Schwanniomyces occidentalis; and filamentousfungi such as, e.g., Neurospora, Penicillium, Tolypocladium, andAspergillus hosts such as A. nidulans and A. niger.

Suitable host cells for the expression of glycosylated antibody arederived from multicellular organisms. Examples of invertebrate cellsinclude plant and insect cells. Numerous baculoviral strains andvariants and corresponding permissive insect host cells from hosts suchas Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedesalbopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyxmori have been identified. A variety of viral strains for transfectionare publicly available, e.g., the L-1 variant of Autographa californicaNPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be usedas the virus herein according to the present invention, particularly fortransfection of Spodoptera frugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,tobacco, lemna, and other plant cells can also be utilized as hosts.

Examples of useful mammalian host cell lines are Chinese hamster ovarycells, including CHOK1 cells (ATCC CCL61), DXB-11, DG-44, and Chinesehamster ovary cells/−DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci.USA 77: 4216 (1980)); monkey kidney CV1 line transformed by SV40 (COS-7,ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subclonedfor growth in suspension culture, (Graham et al., J. Gen Virol. 36: 59,1977); baby hamster kidney cells (BHK, ATCC CCL 10); mouse sertoli cells(TM4, Mather, (Biol. Reprod. 23: 243-251, 1980); monkey kidney cells(CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCCCRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); caninekidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCCCRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (HepG2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells(Mather et al., Annals N.Y Acad. Sci. 383: 44-68 (1982)); MRC 5 cells;FS4 cells; and a human hepatoma line (Hep G2).

Host cells are transformed or transfected with the above-describedexpression or cloning vectors for antibody production and cultured inconventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences. In addition, novel vectors and transfected cell lineswith multiple copies of transcription units separated by a selectivemarker are particularly useful and preferred for the expression ofantibodies that bind the desired antigen.

Host cells containing desired antibody nucleic acid sequences may becultured in a variety of media. Commercially available media such asHam's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) aresuitable for culturing the host cells. In addition, any of the mediadescribed in Ham et al., (Meth. Enz. 58: 44, 1979), Barnes et al., Anal.Biochem. 102: 255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866;4,927,762; 4,560,655; or 5,122,469; WO90103430; WO 87/00195; or U.S.Pat. Re. No. 30,985 may be used as culture media for the host cells. Anyof these media may be supplemented as necessary with hormones and/orother growth factors (such as insulin, transferrin, or epidermal growthfactor), salts (such as sodium chloride, calcium, magnesium, andphosphate), buffers (such as HEPES), nucleotides (such as adenosine andthymidine), antibiotics (such as GENTAMYCIN™ drug), trace elements(defined as inorganic compounds usually present at final concentrationsin the micromolar range), and glucose or an equivalent energy source.Any other necessary supplements may also be included at appropriateconcentrations that would be known to those skilled in the art. Theculture conditions, such as temperature, pH, and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

When using recombinant techniques, the antibody can be producedintracellularly, in the periplasmic space, or directly secreted into themedium, including from microbial cultures. If the antibody is producedintracellularly, as a first step, the particulate debris, either hostcells or lysed fragments, is removed, for example, by centrifugation orultrafiltration. Better et al. (Science 240:1041-43, 1988; ICSU ShortReports 10:105 (1990); and Proc. Natl. Acad. Sci. USA 90:457-461 (1993)describe a procedure for isolating antibodies which are secreted to theperiplasmic space of E. coli. [See also, (Carter et al., Bio/Technology10:163-167 (1992)].

The antibody composition prepared from microbial or mammalian cells canbe purified using, for example, hydroxylapatite chromatography cation oravian exchange chromatography, and affinity chromatography, withaffinity chromatography being the preferred purification technique. Thesuitability of protein A as an affinity ligand depends on the speciesand isotype of any immunoglobulin Fc domain that is present in theantibody. Protein A can be used to purify antibodies that are based onhuman γ1, γ2, or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13, 1983). Protein G is recommended for all mouse isotypes and forhuman γ3 (Guss et al., EMBO J. 5:15671575 (1986)). The matrix to whichthe affinity ligand is attached is most often agarose, but othermatrices are available. Mechanically stable matrices such as controlledpore glass or poly(styrenedivinyl)benzene allow for faster flow ratesand shorter processing times than can be achieved with agarose. Wherethe antibody comprises a C_(H) 3 domain, the Bakerbond ABX™ resin (J. T.Baker, Phillipsburg, N.J.) is useful for purification. Other techniquesfor protein purification such as fractionation on an ion-exchangecolumn, ethanol precipitation, Reverse Phase HPLC, chromatography onsilica, chromatography on heparin SEPHAROSE™ chromatography on an anionor cation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Antibodies of the Invention

The present invention encompasses target specific antibodies that bindinsulin, insulin receptor and/or the insulin/insulin receptor complex,and preferably alter (e.g. increase or decrease) signaling of theinsulin receptor and/or its effect on glucose levels and glucose uptake.In exemplary embodiments, a target specific antibody of the inventioncan comprise a human kappa (κ) or a human lambda (λ) light chain or anamino acid sequence derived therefrom, or a human heavy chain or asequence derived therefrom, or both heavy and light chains together in asingle chain, dimeric, tetrameric or other form. In some embodiments, aheavy chain and a light chain of a target specific immunoglobulin aredifferent amino acid molecules. In other embodiments, the same aminoacid molecule contains a heavy chain variable region and a light chainvariable region of a target specific antibody.

In some embodiments, the amino acid sequence of the anti-target antibodycomprises one or more CDRs of the amino acid sequence of the mature(i.e., missing signal sequence) light chain variable region (V_(L)) ofantibodies in SEQ ID NO: 1-150 or variants thereof. In some embodiments,the V_(L) comprises the amino acid sequence from the beginning of theCDR1 to the end of the CDR3 of the light chain of any one of theforegoing antibodies.

In one embodiment, the target specific antibody comprises a light chainCDR1, CDR2 or CDR3 (LCDR1, LCDR2, LCDR3), each of which areindependently selected from the CDR1, CDR2 and CDR3 regions of anantibody having a light chain variable region comprising the amino acidsequence of the V_(L) region set out in SEQ ID NOs: 1-150. In one aspectthe light chain CDR1 is within residues 24-36, CDR2 is within residues50-56 and CDR3 is within residues 89-101, according to Chothianumbering. A polypeptide of the target specific antibody may comprisethe CDR1, CDR2 and CDR3 regions of an antibody having the amino acidsequence of the V_(L) region selected from the group consisting of SEQID NOs: 1-150.

In some embodiments, the target specific antibody comprises one or moreCDRs of the amino acid sequence of the mature (i.e., missing signalsequence) heavy chain variable region (V_(H)) of antibodies set out inSEQ ID NOs: 151-303 or variants thereof. In some embodiments, the V_(H)comprises the amino acid sequence from the beginning of the CDR1 to theend of the CDR3 of any one of the heavy chain of the foregoingantibodies.

In one embodiment, the target specific antibody comprises a heavy chainCDR1, CDR2 or CDR3 (HCDR1, HCDR2, HCDR3), each of which areindependently selected from the CDR1, CDR2 and CDR3 regions of anantibody having a heavy chain variable region comprising the amino acidsequence of the V_(H) region set out in SEQ ID NOs: 151-303. It isfurther contemplated that a target specific antibody comprises a heavychain CDR1, CDR2 or CDR3, each of which are independently selected fromthe CDR1, CDR2 and CDR3 regions of an antibody having a heavy chainvariable region having the amino acid sequence of the V_(H) region setout in SEQ ID NOs: 151-303. In one aspect the heavy chain CDRs arelocated according to Chothia numbering: CDR1 is within residues 26-35,CDR2 is within residues 50-58 and CDR3 is within residues 95-111 or97-118. A polypeptide of the target specific antibody may comprise theCDR1, CDR2 and CDR3 regions of an antibody having the amino acidsequence of the V_(H) region selected from the group consisting of SEQID NOs: 151-303.

CDRs in Tables 1 and 2 and SEQ ID NO: 1-303 were determined according tothe IMGT system, LeFranc et al IMGT, the INTERNATIONAL IMMUNOGENETICSINFORMATION SYSTEM®, Nucl. Ac. Res. 33 D593-597 (2005).

In another embodiment, the antibody comprises a mature light chainvariable region as disclosed above and a mature heavy chain variableregion as disclosed above, paired as set forth in Table 3. In anotherembodiment, the invention contemplates a purified preparation of amonoclonal antibody, comprising the light chain variable region andheavy chain variable regions of any of antibodies as set forth in SEQ IDNOs: 1-303 and paired as set forth in Table 3.

In exemplary embodiments, the invention contemplates:

a monoclonal antibody that retains any one, two, three, four, five, orsix of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3 of any one of SEQ IDNOs: 151-303 and SEQ ID NOs: 1-150, respectively, optionally includingone or two mutations in any of such CDR(s), e.g., a conservative ornon-conservative substitution, and optionally paired as set forth inTable 3;

a monoclonal antibody that retains all of HCDR1, HCDR2, HCDR3, or theheavy chain variable region of any one of SEQ ID NOs: 151-303,optionally including one or two mutations in any of such CDR(s),optionally further comprising any suitable heavy chain constant region,e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, or IgE, a human sequencethereof, or a hybrid thereof or a human consensus thereof;

a monoclonal antibody that retains all of LCDR1, LCDR2, LCDR3, or thelight chain variable region of any one SEQ ID NOs: 1-150, optionallyincluding one or two mutations in any of such CDR(s), optionally furthercomprising to any suitable light chain constant region, e.g. a kappa orlambda light chain constant region, a human sequence thereof, or ahybrid thereof or a human consensus thereof;

a monoclonal antibody that binds to the same linear or three-dimensionalepitope of INSR as an antibody comprising variable regions set out inSEQ ID NO: 1-303, e.g., as determined through X-ray crystallography orother biophysical or biochemical techniques such as deuterium exchangemass spectrometry, alanine scanning and peptide fragment ELISA;

a monoclonal antibody that competes with an antibody comprising variableregions set out in SEQ ID NO: 1-303 for binding to human INSR by morethan about 75%, more than about 80%, or more than about 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% or 95%.

In some embodiments, the antibody comprises all three light chain CDRs,all three heavy chain CDRs, or all six CDRs of the light and heavychain, paired as set forth in Table 3. In some exemplary embodiments,two light chain CDRs from an antibody may be combined with a third lightchain CDR from a different antibody. Alternatively, a LCDR1 from oneantibody can be combined with a LCDR2 from a different antibody and aLCDR3 from yet another antibody, particularly where the CDRs are highlyhomologous. Similarly, two heavy chain CDRs from an antibody may becombined with a third heavy chain CDR from a different antibody; or aHCDR1 from one antibody can be combined with a HCDR2 from a differentantibody and a HCDR3 from yet another antibody, particularly where theCDRs are highly homologous.

Consensus CDRs may also be used. Any one of the consensus CDRs derivedherein may be combined with two other CDRs from the same chain (e.g.heavy or light) of any of antibodies, e.g. to form a suitable heavy orlight chain variable region.

In some embodiments, an antibody is provided that comprises apolypeptide having an amino acid sequence at least about 65%, 70%, 75%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more identical to the heavy chainvariable region set out in SEQ ID NO: 148-284 and/or an amino acidsequence an amino acid sequence at least about 65%, 70%, 75%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more identical to the light chain variable regionset out in SEQ ID NO: 1-150, the antibody further comprising at leastone, two, three, four, five or all of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2or CDRL3. In some embodiments, the amino acid sequence with percentageidentity to the light chain variable region may comprise one, two orthree of the light chain CDRs. In other embodiments, the amino acidsequence with percentage identity to the heavy chain variable region maycomprise one, two, or three of the heavy chain CDRs.

It is contemplated that the antibodies of the invention may have one, ortwo or more amino acid substitutions in the CDR regions of the antibody,e.g., non-conservative or conservative substitutions.

In a related embodiment, the residues of the framework are altered. Theheavy chain framework regions which can be altered lie within regionsdesignated H-FR1, H-FR2, H-FR3 and H-FR4, which surround the heavy chainCDR residues, and the residues of the light chain framework regionswhich can be altered lie within the regions designated L-FR1, L-FR2,L-FR3 and L-FR4, which surround the light chain CDR residues. An aminoacid within the framework region may be replaced, for example, with anysuitable amino acid identified in a human framework or human consensusframework.

It is further contemplated that the invention provides a purifiedpolypeptide comprising any one of the amino acid sequences of SEQ ID NO:1-150 fused to any one of the amino acid sequences of SEQ ID NO:151-303, optionally paired as the heavy/light chain variable regions setforth in Table 3, or fragments thereof that include at least a portionof SEQ ID NO: 1-150 and SEQ ID NO: 151-303, optionally paired as setforth in Table 3, wherein the polypeptide binds insulin receptor,insulin or the insulin/insulin receptor complex.

In another aspect, the invention provides a purified polypeptidecomprising at least one CDR of a light chain variable region describedherein, wherein the light chain variable region comprises an amino acidsequence at least 90% identical to the LCDR sequences set out in SEQ IDNO: 1-150. In one embodiment, the polypeptide may be 90%, 95%, 96%, 97%,98%, or 99% identical to any one of the LCDRs set out in SEQ ID NO:1-150. In a further aspect, the invention provides a purifiedpolypeptide comprising at least one CDR of a heavy chain variable regiondescribed herein, wherein the heavy chain variable region comprises anamino acid sequence at least 90% identical to the HCDR sequences set outin SEQ ID NO: 151-303. In one embodiment, the polypeptide may be 90%,95%, 96%, 97%, 98%, or 99% identical to any one of the HCDRs set out inSEQ ID NO: 151-303.

It is further contemplated that the CDR of the antibody heavy and lightchains comprise variant amino acid sequences which may improve antibodybinding affinity and are derived through, for example, affinitymaturation. In one aspect it is contemplated that an antibody of theinvention comprises a heavy chain HCDR2 sequence having about 35%identity to a HCDR2 of a parent antibody sequence set out in SEQ ID NOs:151-303. In a related aspect it is contemplated that an antibody of theinvention comprises a heavy chain HCDR3 sequence having about 50%identity to a HCDR3 of a parent antibody sequence set out in SEQ ID NOs:151-303.

In one embodiment the invention provides antigen-binding compounds,including functional fragments, having a variable region amino acidsequence set forth in any one of SEQ ID NOs: 1-150 and 151-303. In arelated embodiment, an aforementioned antigen binding compound isselected from the group consisting of a fully assembled tetramericantibody, a polyclonal antibody, a monoclonal antibody including a HUMANENGINEERED™ antibody; a humanized antibody; a human antibody; a chimericantibody; a multispecific antibody, an antibody fragment, Fab, F(ab′)₂;Fv; scFv or single-chain antibody fragment; a diabody; triabody,tetrabody, minibody, linear antibody; chelating recombinant antibody, atribody or bibody, an intrabody, a nanobody, a small modularimmunopharmaceutical (SMIP), a binding-domain immunoglobulin fusionprotein, a camelized antibody, a V_(HH) containing antibody, or avariant or derivative of any one of these antibodies, that comprise oneor more CDR sequences of the invention and exhibit the desiredbiological activity. The antigen binding compounds of the inventionpreferably retain binding affinity of 10⁻⁵, 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹10⁻¹⁰, 10⁻¹¹ M or less as measured by surface plasmon resonance.

In one aspect, the antibodies of the invention comprise a heavy chainvariable region or light chain variable region as set out in amino acidsequences SEQ ID NO: 151-303 and SEQ ID NO: 1-150, respectively, aspaired in Table 3. It is further contemplated that the antibodies maycomprise all or part of the antibodies set out in the above amino acidsequences. In one embodiment, the antibodies comprise at least one ofCDR1, CDR2, or CDR3 of the heavy chain of SEQ ID NOs: 151-303, or atleast one of CDR1, CDR2 or CDR3 of the light chain of SEQ ID NOs: 1-150,as paired in Table 3.

In one embodiment, the heavy chain comprises an amino acid sequenceidentified as a heavy chain CDR3 sequence. Such a “heavy chain CDR3sequence” (HCDR3) includes an amino acid sequence identified as a heavychain CDR3 sequence set out in Table 2 and SEQ ID NOs: 151-303.Alternatively, the HCDR3 sequence comprises an amino acid sequence thatcontains one or more amino acid changes compared to any HCDR3 amino acidsequence identified in Table 2, i.e., a substitution, insertion ordeletion. Preferable substitutions include a substitution to an aminoacid at the corresponding position within another HCDR3 of Table 2.Alternatively, the HCDR3 sequence may comprise a consensus amino acidsequence of the HCDR3 described herein.

Alternatively, the heavy chain comprising a HCDR3 sequence of theinvention described above may further comprise a “heavy chain CDR2sequence” (HCDR2) of the invention, which includes any of the amino acidsequences identified as an HCDR2 in SEQ ID NO: 151-303 and Table 2,amino acid sequences that contain one or more amino acid changescompared to any HCDR2 identified in SEQ ID NO: 151-303 and Table 2,preferably a substitution to an amino acid at the corresponding positionwithin another HCDR2 of Table 2, or a consensus sequence of the HCDR2described herein.

The heavy chain comprising a heavy chain CDR3 sequence of the inventiondescribed above may also comprise both (a) a heavy chain CDR1 sequenceof the invention described above and (b) a heavy chain CDR2 sequence ofthe invention described above.

One aspect of the invention provides an antibody that binds targetantigen comprising a heavy chain that comprises any one, two, and/orthree of the heavy chain CDR sequences of the invention described below.

Any of the heavy chain CDR sequences described above may also includeamino acids added to either end of the CDRs. Preparation of variants andderivatives of antibodies and antigen-binding compounds of theinvention, including affinity maturation or preparation of variants orderivatives containing amino acid analogs, is described in furtherdetail herein. Exemplary variants include those containing aconservative or non-conservative substitution of a corresponding aminoacid within the amino acid sequence, or a replacement of an amino acidwith a corresponding amino acid of a human antibody sequence.

Antibodies comprising any one of the heavy chains described above mayfurther comprise a light chain, preferably a light chain that binds totarget antigen, and most preferably a light chain comprising light chainCDR sequences of the invention described below.

Another aspect of the invention provides an antibody that binds targetantigen comprising a light chain that comprises any one, two, and/orthree of the light chain CDR sequences of the invention described below.

Preferably the light chain comprises an amino acid sequence identifiedas a light chain CDR3 sequence. Such a “light chain CDR3 sequence”(LCDR3) includes an amino acid sequence identified as a light chain CDR3sequence in Table 1 and within SEQ ID NOs: 1-150. Alternatively, thelight chain CDR3 sequence comprises an amino acid sequence that containsone or more amino acid changes compared to any light chain CDR3 aminoacid sequence identified in Table 1, i.e. a substitution, insertion ordeletion. Preferable substitutions include a substitution to an aminoacid at the corresponding position within another light chain CDR3 ofTable 1. Alternatively, the light chain CDR3 sequence may comprise aconsensus amino acid sequence of light chain CDR3 shown in Table 1.

The light chain comprising a light chain CDR3 sequence of the inventiondescribed above may further comprise a “light chain CDR1 sequence” ofthe invention, which includes any of the amino acid sequences identifiedas a light chain CDR1 in SEQ ID NO: 1-150 or Table 1, amino acidsequences that contain one or more amino acid changes compared to anylight chain CDR1 identified in SEQ ID NO: 1-150 or Table 1, preferably asubstitution to an amino acid at the corresponding position withinanother light chain CDR1 of Table 1, or a consensus sequence of lightchain CDR1 described herein.

Alternatively, the light chain comprising a light chain CDR3 sequence ofthe invention described above may further comprise a “light chain CDR2sequence” of the invention, which includes any of the amino acidsequences identified as a light chain CDR2 in SEQ ID NO: 1-150 or Table1, amino acid sequences that contain one or more amino acid changescompared to any light chain CDR2 identified in Table 1, preferably asubstitution to an amino acid at the corresponding position withinanother light chain CDR2 of SEQ ID NO: 1-150 or Table 1, or a consensussequence of light chain CDR2 shown in Table 1.

In a related aspect, the invention contemplates a purified polypeptidecomprising at least one HCDR of SEQ ID NO: 151-303 or LCDR of SEQ ID NO:1-150, wherein the framework regions of the heavy chain variable regionand the framework regions of the light chain variable region compriseframework regions from a human antibody. In another embodiment, theframework regions of the heavy chain variable region and the frameworkregions of the light chain variable region are chemically altered byamino acid substitution to be more homologous to a human antibodysequence. For example, within each heavy chain framework region(H-FR1-4) it is contemplated that at least one, at least two, at leastthree, at least four, at least five, or at least six native frameworkregion residues of the murine heavy chain variable region have beenaltered by amino acid substitution, and wherein within each light chainframework region (L-FR1-4), at least one, at least two, at least three,at least four, at least five or at least six native framework residuesof the murine light chain variable region have been altered by aminoacid substitution.

The light chain comprising a light chain CDR3 sequence of the inventiondescribed above may also comprise both (a) a light chain CDR1 sequenceof the invention described above and (b) a light chain CDR2 sequence ofthe invention described above.

Antibodies comprising any one of the light chain variable regionsdescribed above may further comprise a heavy chain variable region,optionally paired as described in Table 3, preferably a heavy chainvariable region that binds to target antigen, and most preferably aheavy chain variable region comprising heavy chain CDR sequences of theinvention described above.

In one aspect, the antibody binds to insulin receptor or a complexcomprising insulin and insulin receptor with an equilibrium dissociationconstant K_(D) of 10⁻⁵M or less that is capable of strengthening thebinding affinity between insulin and insulin receptor by about 5-fold to200-fold. In one embodiment, the antibody is a positive modulatorantibody, e.g., that strengthens the binding affinity between insulinand insulin receptor. In some embodiments, the positive modulatorantibody includes, but is not limited to Ab006, Ab030, Ab004, Ab013,Ab009, Ab007, Ab011, Ab001, Ab012, Ab010, Ab003, Ab008, Ab002, Ab005,Ab076, Ab077, Ab079, Ab080, Ab083, Ab059, Ab078, Ab085 or anypolypeptide comprising one or more of the CDRs corresponding to any oneof the above antibodies as set out in Tables 1 and 2, or in an antibodyvariable region set out in SEQ ID NOs: 76, 80, 101, 128, 132 and SEQ IDNOs: 291, 196, 239, 267, 271.

In further embodiments, the positive modulator antibody binds to insulinreceptor, the insulin/insulin receptor complex, or binds both insulinreceptor and the insulin/insulin receptor complex. In a relatedembodiment, the positive modulator antibody that binds to insulinreceptor or insulin/insulin receptor complex, or both, includes, but isnot limited to Ab006, Ab030, Ab004, Ab013, Ab009, Ab007, Ab011, Ab001,Ab012, Ab010, Ab003, Ab008, Ab002, Ab005, Ab076, Ab077, Ab079, Ab080,Ab083 or any polypeptide comprising one or more of the CDRscorresponding to any one of the above antibodies as set out in Tables 1and 2, or in an antibody variable region set out in SEQ ID NOs: 76, 80,101 and SEQ ID NOs: 291, 196 and 239.

In a further embodiment, the positive modulator antibody binds to theinsulin/insulin receptor complex but not detectably to uncomplexedinsulin receptor. In a related embodiment, the positive modulatorantibody that binds to the insulin/insulin receptor complex includes,but is not limited to, Ab059, Ab078, Ab085 or any polypeptide comprisingone or more of the CDRs corresponding to any one of the above antibodiesas set out in Tables 1 and 2, or in an antibody variable region set outin SEQ ID NOs: 128, 132 and SEQ ID NOs: 267 and 271.

In a related aspect, the antibody is an agonist antibody. In oneembodiment, the antibody is an agonist antibody that binds to insulinreceptor with an affinity of 10⁻⁵, 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹ 10⁻¹⁰, 10⁻¹¹ Mor less, optionally that exhibits maximal agonist activity that is20%-100% that of insulin's maximal agonist activity when measured inpAKT assay. In a further embodiment, the antibody is an allostericagonist antibody that binds to insulin receptor with an affinity of10⁻⁵, 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹ 10⁻¹⁰, 10⁻¹¹ M or less and (a) exhibitsmaximal agonist activity that is 20%-80% that of insulin's maximalagonist activity when measured in pAKT assay, (b) when present does notalter the EC50 of insulin for INSR by more than 2-fold, and (c) whenpresent does not alter the K_(D) of insulin for INSR by more than2-fold.

In certain embodiments, the agonist antibody includes, but is notlimited to, Ab021, Ab029, Ab022, Ab017, Ab023, Ab024, Ab025, Ab026,Ab031, Ab035, Ab027, Ab036, Ab037, Ab028, Ab038, Ab039, Ab040, Ab041,Ab042, Ab032, Ab043, Ab044, Ab045, Ab046, Ab047, Ab018, Ab033, Ab048,Ab014, Ab015, Ab049, Ab034, Ab051, Ab053, Ab054, Ab056, Ab058, Ab062,Ab064, Ab066, Ab067, Ab068, Ab086, Ab069, Ab071, Ab073, Ab075, Ab082,Ab084 or any polypeptide comprising one or more of the CDR correspondingto any one of the above antibodies as set out in Tables 1 and 2, or inan antibody variable region set out in SEQ ID NOs: 7, 113, 114, 124,126, 130 and SEQ ID NOs: 164, 252, 253, 263, 265 and 269.

In a further aspect, the antibody binds to insulin receptor or a complexcomprising insulin and insulin receptor with an equilibrium dissociationconstant K_(D) of 10⁻⁵M or less that is capable of weakening the bindingaffinity between insulin and insulin receptor by at least about 3-fold,optionally up to 1000-fold. In one embodiment, the antibody is anegative modulator antibody that weakens the binding affinity betweeninsulin and the insulin receptor. In a related embodiment, the negativemodulator antibody includes, but is not limited to the followingantibodies: Ab087, Ab019, Ab088, Ab089, Ab020, Ab050, Ab052, Ab055,Ab057, Ab061, Ab063, Ab065, Ab070, Ab072, Ab074 and Ab081.

In a further aspect, the antibody is an antibody that competes with anyof the antibodies described herein for binding to the insulin receptoror insulin/insulin receptor complex. In certain embodiments, theantibody exhibits partial competition. In a related embodiment, partialcompetition is competition of about 30% to 70%, about 30% to 80%, orabout 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80%. In someembodiments, the antibody exhibits complete competition. In oneembodiment, complete competition is competition of greater than 70%,75%, 80%, 85%, 90%, 95% or 100%. Exemplary assays for measuring antibodycompetition include, but are not limited to, receptor loading assays andepitope binning assays as described herein and in the art.

In one embodiment, the antibody exhibits greater than or equal to 70%competition with any one, two, three or all antibodies selected from thegroup consisting of Ab079, Ab076, Ab083, Ab080, Ab062, Ab020, Ab019,Ab088, and Ab089, and optionally, exhibit greater than or equal to 30%competition with any one, two, three or all antibodies selected from thegroup consisting of Ab086, Ab064, Ab001, and Ab018. In a furtherembodiment, the antibody optionally does not compete with one or more ofAb062 and Ab086. In certain embodiments, the antibody binds to bothhuman and murine insulin receptor or complex.

In a further embodiment, the antibody that competes with an antibodydescribed herein exhibits greater than or equal to 70% competition withany one, two, three or all antibodies selected from the group consistingof Ab040, Ab062, Ab030, Ab001, and Ab018, and optionally exhibit greaterthan or equal to 30% competition with any one, two, three or allantibodies selected from the group consisting of AB037, Ab078, AB083,AB080, and AB085. In a related embodiment, the antibody does not competewith Ab053, Ab064, 83-7, Ab019, Ab088, and Ab089. Optionally, theantibody binds to human and murine insulin receptor or complex.

In a further embodiment, the antibody that competes with an antibodydescribed herein exhibits greater than or equal to 70% competition withany one, two, three or all antibodies selected from the group consistingof Ab030, Ab037, Ab053, Ab001, Ab018, Ab064, Ab040, and optionallyexhibit greater than or equal to 30% competition with any one, two,three or all antibodies selected from the group consisting of Ab085 andAb086. Optionally, the antibody exhibits no competition with Ab079,Ab076 and Ab088, and optionally binds to both human and murine insulinreceptor or complex.

In a further embodiment, the antibody that competes with an antibodydescribed herein that exhibits greater than or equal to 70% competitionwith any one, two, three or all antibodies selected from the groupconsisting of Ab064, Ab062, Ab085, and Ab078, and optionally exhibits nocompetition with Ab077, Ab001, Ab018, Ab030, Ab037, Ab079, Ab076, Ab083,Ab019, Ab088, Ab089, and Ab040. Optionally, the antibody binds bothhuman and murine insulin receptor or complex.

In a further embodiment, the antibody that competes with an antibodydescribed herein exhibits greater than or equal to 70% competition withany one, two, three or all antibodies selected from the group consistingof Ab079, Ab076, Ab083, Ab080, Ab062, Ab020, Ab019, Ab088, Ab089.Optionally, the antibody does not exhibit competition with Ab062, Ab086,Ab001, Ab018, Ab030, Ab037, Ab064; and optionally, the antibody is humanreactive only, and does not bind murine insulin receptor or complex.

In a further embodiment, the antibody shows greater than or equal to 30%competition with any antibody. Optionally, the antibody shows greaterthan or equal to 30% competition with Ab061, and optionally has lessthan 30% competition with Ab019 and Ab074, optionally shows nocompetition with Ab088. Optionally, the antibody binds with both humanand murine receptor or complex.

In yet another embodiment, the antibody comprises a heavy chain variableregion selected from the group consisting of SEQ ID NOs: 281, 278, 277,209, 275, 223, 284, 276, and 236 and a light chain variable regionselected from the group consisting of SEQ ID NOs: 141, 138, 137, 35,135, 57, 144, 136, and 98.

In yet another embodiment, the antibody comprises a heavy chain variableregion selected from the group consisting of SEQ ID NOs: 195, 220, 303,197, 208, 243, 245 and 251 and a light chain variable region selectedfrom the group consisting of SEQ ID NOs: 77, 50, 90, 84, 34, 104, 106and 112.

In yet another embodiment, the antibody comprises a heavy chain variableregion selected from the group consisting of SEQ ID NOs: 241, 279, 258,155, and 228 and a light chain variable region selected from the groupconsisting of SEQ ID NOs: 103, 139, 119, 8, and 89.

Antibody Nucleic Acids of the Invention

The present invention also encompasses nucleic acid molecules encodingtarget specific antibodies as described above. In some embodiments,different nucleic acid molecules encode a heavy chain variable regionand a light chain variable region of a target specific antibody. Inother embodiments, the same nucleic acid molecule encodes a heavy chainand a light chain variable regions of a target specific antibody. In oneembodiment, the nucleic acid encodes a target specific antibody of theinvention.

In one aspect, a nucleic acid molecule of the invention comprises anucleotide sequence that encodes the V_(L) amino acid sequence set outin any one of SEQ ID NOs: 1-150 or a portion thereof. In a relatedaspect, the V_(L) amino acid sequence is a consensus sequence. In someembodiments, the nucleic acid encodes the amino acid sequence of thelight chain CDRs of said antibody. In some embodiments, said portion isa contiguous portion comprising CDR1-CDR3. In one embodiment, saidportion comprises at least one, two or three of a light chain CDR1,CDR2, or CDR3 region.

In some embodiments, the nucleic acid molecule encodes a V_(L) aminoacid sequence that is at least 60, 65, 70, 75, 80, 85, 90, 91, 92, 93,94, 95, 96 97, 98 or 99% identical to a V_(L) amino acid sequence setout in SEQ ID NOs: 1-150. Nucleic acid molecules of the inventioninclude nucleic acids that hybridize under highly stringent conditions.

It is further contemplated that a nucleic acid molecule of the inventioncomprises a nucleotide sequence that encodes the V_(H) amino acidsequence of any one of SEQ ID NO: 151-303, or a portion thereof. In arelated aspect, the V_(H) amino acid sequence is a consensus sequence.In some embodiments, the nucleic acid encodes the amino acid sequence ofthe heavy chain CDRs of said antibody. In some embodiments, said portionis a contiguous portion comprising heavy chain CDR1-CDR3. In oneembodiment, said portion comprises at least one, two or three of a heavychain CDR1, CDR2, or CDR3 region.

In some embodiments, the nucleic acid molecule encodes a V_(H) aminoacid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98% or 99% identical to a V_(H) amino acid sequence set out in SEQ IDNOs: 151-303. Nucleic acid molecules of the invention include nucleicacids that hybridize under highly stringent conditions.

It is further contemplated that the nucleic acids of the inventionencode a full-length light chain or heavy chain of an antibodycomprising a heavy chain or light chain variable region set out in SEQID NOs:1-303 and optionally paired as described in Table 3, wherein afull-length light chain or full-length heavy chain comprises a lightchain constant region or a heavy chain constant region, respectively.

The invention further contemplates nucleic acids encoding antibodyvariants and polypeptides comprising antigen binding regions of theinvention as described above.

Methods of preparing and isolating polynucleotide encoding antibodies ofthe invention are well-known to those of skill in the art. Apolynucleotide according to the invention can be joined to any of avariety of other nucleotide sequences by well-established recombinantDNA techniques (see Sambrook et al., (2d Ed.; 1989) Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y.). Useful nucleotide sequences for joining to polypeptides includean assortment of vectors, e.g., plasmids, cosmids, lambda phagederivatives, phagemids, and the like, that are well known in the art.Accordingly, the invention also provides a vector including apolynucleotide of the invention and a host cell containing thepolynucleotide. In general, the vector contains an origin of replicationfunctional in at least one organism, convenient restriction endonucleasesites, and a selectable marker for the host cell. Vectors according tothe invention include expression vectors, replication vectors, probegeneration vectors, sequencing vectors, and retroviral vectors. A hostcell according to the invention can be a prokaryotic or eukaryotic celland can be a unicellular organism or part of a multicellular organism.Large numbers of suitable vectors and promoters are known to those ofskill in the art and are commercially available for generating therecombinant constructs of the present invention.

A variety of expression vector/host systems may be utilized to containand express the coding sequence. These include, but are not limited to,microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, phagemid, or cosmid DNA expression vectors;yeast transformed with yeast expression vectors; insect cell systemsinfected with viral expression vectors (e.g., baculovirus); plant cellsystems transfected with virus expression vectors (e.g., CauliflowerMosaic Virus, CaMV; Tobacco Mosaic Virus, TMV) or transformed withbacterial expression vectors (e.g., Ti or pBR322 plasmid); or evenanimal cell systems. Mammalian cells that are useful in recombinantprotein productions include, but are not limited to, VERO cells, HeLacells, Chinese hamster ovary (CHO) cells, COS cells (such as COS-7),WI38, BHK, HepG2, 3T3, RIN, MDCK, A549, PC12, K562 and HEK 293 cells.

Polynucleotide variants and antibody fragments may be readily generatedby a worker of skill to encode biologically active fragments, variants,or mutants of the naturally occurring antibody molecule that possess thesame or similar biological activity to the naturally occurring antibody.This may be done by PCR techniques, cutting and digestion of DNAencoding the antibody heavy and light chain regions, and the like. Forexample, point mutagenesis, using PCR and other techniques well-known inthe art, may be employed to identify with particularity which amino acidresidues are important in particular activities associated with antibodyactivity. Thus, one of skill in the art will be able to generate singlebase changes in the DNA strand to result in an altered codon and amissense mutation.

Antibody Fragments

Antibody fragments comprise a portion of an intact full length antibody,preferably an antigen binding or variable region of the intact antibody.Examples of antibody fragments include Fab, Fab′, F(ab′)₂, and Fvfragments; diabodies; linear antibodies; single-chain antibody molecules(e.g., scFv); multispecific antibody fragments such as bispecific,trispecific, etc. antibodies (e.g., diabodies, triabodies, tetrabodies);minibody; chelating recombinant antibody; tribodies or bibodies;intrabodies; nanobodies; small modular immunopharmaceuticals (SMIP),binding-domain immunoglobulin fusion proteins; camelized antibodies;V_(HH) containing antibodies; and other polypeptides formed fromantibody fragments. See for example Holliger & Hudson (Nat. Biotech.23(9) 1126-36 (2005))

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, monovalent fragments consisting ofthe V_(L), V_(H), C_(L) and C_(H) domains each with a singleantigen-binding site, and a residual “Fc” fragment, whose name reflectsits ability to crystallize readily. Pepsin treatment yields a F(ab′)₂fragment, a bivalent fragment comprising two Fab fragments linked by adisulfide bridge at the hinge region, that has two “Single-chain Fv” or“scFv” antibody fragments comprise the V_(H) and V_(L) domains ofantibody, wherein these domains are present in a single polypeptidechain. Preferably, the Fv polypeptide further comprises a polypeptidelinker between the V_(H) and V_(L) domains that enables the Fv to formthe desired structure for antigen binding, resulting in a single-chainantibody (scFv), in which a V_(L) and V_(H) region are paired to form amonovalent molecule via a synthetic linker that enables them to be madeas a single protein chain (Bird et al., Science 242:423-426, 1988, andHuston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988). For areview of scFv see Pluckthun, in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994). An Fd fragment consists of the V_(H) andC_(H)1 domains.

Additional antibody fragments include a domain antibody (dAb) fragment(Ward et al., Nature 341:544-546, 1989) which consists of a V_(H)domain. Diabodies are bivalent antibodies in which V_(H) and V_(L)domains are expressed on a single polypeptide chain, but using a linkerthat is too short to allow for pairing between the two domains on thesame chain, thereby forcing the domains to pair with complementarydomains of another chain and creating two antigen binding sites (seee.g., EP 404,097; WO 93/11161; Holliger et al., Proc. Natl. Acad. Sci.USA 90:6444-6448, 1993, and Poljak et al., Structure 2:1121-1123, 1994).Diabodies can be bispecific or monospecific.

Functional heavy-chain antibodies devoid of light chains are naturallyoccurring in nurse sharks (Greenberg et al., Nature 374:168-73, 1995),wobbegong sharks (Nuttall et al., Mol Immunol. 38:313-26, 2001) andCamelidae (Hamers-Casterman et al., Nature 363: 446-8, 1993; Nguyen etal., J. Mol. Biol. 275: 413, 1998), such as camels, dromedaries, alpacasand llamas. The antigen-binding site is reduced to a single domain, theVHH domain, in these animals. These antibodies form antigen-bindingregions using only heavy chain variable region, i.e., these functionalantibodies are homodimers of heavy chains only having the structure H₂L₂(referred to as “heavy-chain antibodies” or “HCAbs”). Camelid V_(HH)reportedly recombines with IgG2 and IgG3 constant regions that containhinge, CH2, and CH3 domains and lack a CH1 domain (Hamers-Casterman etal., supra). For example, llama IgG1 is a conventional (H₂L₂) antibodyisotype in which V_(H) recombines with a constant region that containshinge, CH1, CH2 and CH3 domains, whereas the llama IgG2 and IgG3 areheavy chain-only isotypes that lack CH1 domains and that contain nolight chains. Camelid V_(HH) domains have been found to bind to antigenwith high affinity (Desmyter et al., J. Biol. Chem. 276:26285-90, 2001)and possess high stability in solution (Ewert et al., Biochemistry41:3628-36, 2002). Classical V_(H)-only fragments are difficult toproduce in soluble form, but improvements in solubility and specificbinding can be obtained when framework residues are altered to be moreVH_(H)-like. (See, e.g., Reichman, et al., J Immunol Methods 1999,231:25-38.) Methods for generating antibodies having camelid heavychains are described in, for example, in U.S. Patent Publication Nos.20050136049 and 20050037421.

The variable domain of an antibody heavy-chain is has a molecular massof 15 kDa, and is referred to as a nanobody (Cortez-Retamozo et al.,Cancer Research 64:2853-57, 2004). A nanobody library may be generatedfrom an immunized dromedary as described in Conrath et al., (AntimicrobAgents Chemother 45: 2807-12, 2001) or using recombinant methods asdescribed in Revets et al, Expert Opin. Biol. Ther. 5(1): 111-24 (2005).

Production of bispecific Fab-scFv (“bibody”) and trispecificFab-(scFv)(2) (“tribody”) are described in Schoonjans et al. (J Immunol.165:7050-57, 2000) and Willems et al. (J Chromatogr B Analyt TechnolBiomed Life Sci. 786:161-76, 2003). For bibodies or tribodies, a scFvmolecule is fused to one or both of the VL-CL (L) and VH-CH₁ (Fd)chains, e.g., to produce a tribody two scFvs are fused to C-term of Fabwhile in a bibody one scFv is fused to C-term of Fab.

A “minibody” consisting of scFv fused to CH3 via a peptide linker(hingeless) or via an IgG hinge has been described in Olafsen, et al.,Protein Eng Des Sel. 2004 April; 17(4):315-23.

Intrabodies are single chain antibodies which demonstrate intracellularexpression and can manipulate intracellular protein function (Biocca, etal., EMBO J. 9:101-108, 1990; Colby et al., Proc Natl Acad Sci USA.101:17616-21, 2004). Intrabodies, which comprise cell signal sequenceswhich retain the antibody construct in intracellular regions, may beproduced as described in Mhashilkar et al (EMBO J 14:1542-51, 1995) andWheeler et al. (FASEB J. 17:1733-5. 2003). Transbodies arecell-permeable antibodies in which a protein transduction domain (PTD)is fused with single chain variable fragment (scFv) antibodies Heng etal., (Med Hypotheses. 64:1105-8, 2005).

Further contemplated are antibodies that are SMIPs or binding domainimmunoglobulin fusion proteins specific for an antigen. These constructsare single-chain polypeptides comprising antigen binding domains fusedto immunoglobulin domains necessary to carry out antibody effectorfunctions. See e.g., WO03/041600, U.S. Patent publication 20030133939and US Patent Publication 20030118592.

One or more CDRs may be incorporated into a molecule either covalentlyor noncovalently to make it an immunoadhesin. An immunoadhesin mayincorporate the CDR(s) as part of a larger polypeptide chain, maycovalently link the CDR(s) to another polypeptide chain, or mayincorporate the CDR(s) noncovalently. The CDRs permit the immunoadhesinto specifically bind to a particular antigen of interest.

In yet another embodiment, the antibody or antigen-binding compoundcomprises a constant region and one or more heavy and light chainvariable framework regions of a human antibody sequence. In a relatedembodiment, the antibody comprises a modified or unmodified constantregion of a human IgG1, IgG2, IgG3 or IgG4.

Alternatively, antibody fragments may be fused to a protein scaffold.Libraries of protein scaffolds include, but are not limited to,Adnectins, Affibodies, Anticalins, DARPins, engineered Kunitz-typeinhibitors, tetranectins, A-domain proteins, lipocalins, repeat proteinssuch as ankyrin repeat proteins, immunity proteins, α2p8 peptide, insectdefensin A, PDZ domains, charybdotoxins, PHD fingers, TEM-1 β-lactamase,fibronectin type III domains, CTLA-4, T-cell resptors, knottins,neocarzinostatin, carbohydrate binding module 4-2, green fluorescentprotein, thioredoxin (Gebauer & Skerra, Curr. Opin. Chem. Biol.13:245-55 (2009); Gill & Damle, Curr. Opin. Biotech 17: 653-58 (2006);Hosse et al, Protein Sci. 15:14-27 (2006); Skerra, Curr. Opin. Biotech18: 295-3-4 (2007)).

Thus, a variety of compositions comprising one, two, and/or three CDRsof a heavy chain variable region or a light chain variable region of anantibody may be generated by techniques known in the art.

Multispecific Antibodies

In some embodiments, it may be desirable to generate multispecific (e.g.bispecific) antibodies of the invention having binding specificities forat least two different epitopes of the same or different molecules.Exemplary bispecific antibodies may bind to two different epitopes ofthe antigen. Alternatively, an antigen-specific antibody arm may becombined with an arm which binds to a cell surface molecule, such as aT-cell receptor molecule (e.g., CD2 or CD3), or Fc receptors for IgG(FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16) so as tofocus cellular defense mechanisms to the desired antigen. Bispecificantibodies may also be used to localize cytotoxic agents to cells whichexpress or take up the desired antigen. These antibodies possess anantigen-binding arm and an arm which binds the cytotoxic agent (e.g.,saporin, anti-interferon-60, vinca alkaloid, ricin A chain, methotrexateor radioactive isotope hapten). Bispecific antibodies can be prepared asfull length antibodies or antibody fragments (e.g., F(ab′)2 bispecificantibodies).

According to another approach for making bispecific antibodies, theinterface between a pair of antibody molecules can be engineered tomaximize the percentage of heterodimers which are recovered fromrecombinant cell culture. The preferred interface comprises at least apart of the C_(H)3 domain of an antibody constant domain. In thismethod, one or more small amino acid side chains from the interface ofthe first antibody molecule are replaced with larger side chains (e.g.,tyrosine or tryptophan). Compensatory “cavities” of identical or similarsize to the large side chain(s) are created on the interface of thesecond antibody molecule by replacing large amino acid side chains withsmaller ones (e.g., alanine or threonine). This provides a mechanism forincreasing the yield of the heterodimer over other unwanted end-productssuch as homodimers. See WO96/27011 published Sep. 6, 1996.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Heteroconjugateantibodies may be made using any convenient cross-linking methods.Suitable cross-linking agents are well known in the art, and aredisclosed in U.S. Pat. No. 4,676,980, along with a number ofcross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,(Science 229:81-83, 1985) describe a procedure wherein intact antibodiesare proteolytically cleaved to generate F(ab′)₂ fragments. Thesefragments are reduced in the presence of the dithiol complexing agentsodium arsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes. In yet afurther embodiment, Fab′-SH fragments directly recovered from E. colican be chemically coupled in vitro to form bispecific antibodies.(Shalaby et al., J. Exp. Med. 175:217-225 (1992))

Shalaby et al., J. Exp. Med. 175:217-225 (1992) describe the productionof a fully humanized bispecific antibody F(ab′)₂ molecule. Each Fab′fragment was separately secreted from E. coli and subjected to directedchemical coupling in vitro to form the bispecfic antibody. Thebispecific antibody thus formed was able to bind to cells overexpressingthe HER2 receptor and normal human T cells, as well as trigger the lyticactivity of human cytotoxic lymphocytes against human breast tumorantigens.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. (Kostelny et al., J. Immunol. 148:1547-1553, 1992). Theleucine zipper peptides from the Fos and Jun proteins were linked to theFab′ portions of two different antibodies by gene fusion. The antibodyhomodimers were reduced at the hinge region to form monomers and thenre-oxidized to form the antibody heterodimers. This method can also beutilized for the production of antibody homodimers. The “diabody”technology described by Holliger et al. (Proc. Natl. Acad. Sci. USA90:6444-48, 1993) has provided an alternative mechanism for makingbispecific antibody fragments.

The fragments comprise a heavy chain variable region (V_(H)) connectedto a light-chain variable region (V_(L)) by a linker which is too shortto allow pairing between the two domains on the same chain. Accordingly,the V_(H) and V_(L) domains of one fragment are forced to pair with thecomplementary V_(L) and V_(H) domains of another fragment, therebyforming two antigen-binding sites. Another strategy for makingbispecific antibody fragments by the use of single-chain Fv (scFv)dimers has also been reported. See Gruber et al., J. Immunol. 152: 5368(1994).

Alternatively, the bispecific antibody may be a “linear antibody”produced as described in Zapata et al. Protein Eng. 8:1057-62 (1995)Linear antibodies comprise a pair of tandem Fd segments(V_(H)-C_(H)1-V_(H)-C_(H)1) which form a pair of antigen bindingregions. Linear antibodies can be bispecific or monospecific.

In a further embodiment, the bispecific antibody may be a chelatingrecombinant antibody (CRAb). A chelating recombinant antibody recognizesadjacent and non-overlapping epitopes of the antigen, and is flexibleenough to bind to both epitopes simultaneously (Neri et al., J Mol Biol.246:367-73, 1995).

Antibodies with more than two valencies are also contemplated. Forexample, trispecific antibodies can be prepared. (Tutt et al., J.Immunol. 147:60, 1991).

Chimeric and Humanized Antibodies

Because chimeric or humanized antibodies are less immunogenic in humansthan the parental mouse monoclonal antibodies, they can be used for thetreatment of humans with far less risk of anaphylaxis.

Chimeric monoclonal antibodies, in which the variable Ig domains of amouse monoclonal antibody are fused to human constant Ig domains, can begenerated using standard procedures known in the art (See Morrison etal., Proc. Natl. Acad. Sci. USA 81, 6841-6855 (1984); and, Boulianne etal, Nature 312, 643-646, (1984)). Although some chimeric monoclonalantibodies have proved less immunogenic in humans, the mouse variable Igdomains can still lead to a significant human anti-mouse response.

Humanized antibodies may be achieved by a variety of methods including,for example: (1) grafting the non-human complementarity determiningregions (CDRs) onto a human framework and constant region (a processreferred to in the art as humanizing through “CDR grafting”) (2)transplanting the entire non-human variable domains, but “cloaking” themwith a human-like surface by replacement of surface residues (a processreferred to in the art as “veneering”), or, alternatively, (3)substituting human amino acids at positions determined to be unlikely toadversely effect either antigen binding or protein folding, but likelyto reduce immunogenicity in a human environment (a process referred toin the art as HUMAN ENGINEERING™). In the present invention, humanizedantibodies will include both “humanized”, “veneered” and “HUMANENGINEERED™” antibodies. These methods are disclosed in, e.g., Jones etal., Nature 321:522 525 (1986); Morrison et al., Proc. Natl. Acad. Sci.,U.S.A., 81:6851-6855 (1984); Morrison and Oi, Adv. Immunol., 44:65-92(1988); Verhoeyer et al., Science 239:1534-1536 (1988); Padlan, Molec.Immun. 28:489-498 (1991); Padlan, Molec. Immunol. 31:169-217 (1994);Kettleborough et al., Protein Eng. 4:773-783 (1991); Studnicka et al.U.S. Pat. No. 5,766,886; Studnicka et al., (Protein Eng 7: 805-814,1994) each of which is incorporated herein by reference.

Human Antibodies from Transgenic Animals

Human antibodies to antigen can also be produced using transgenicanimals that have no endogenous immunoglobulin production and areengineered to contain human immunoglobulin loci. For example, WO98/24893 discloses transgenic animals having a human Ig locus whereinthe animals do not produce functional endogenous immunoglobulins due tothe inactivation of endogenous heavy and light chain loci. WO 91/00906also discloses transgenic non-primate mammalian hosts capable ofmounting an immune response to an immunogen, wherein the antibodies haveprimate constant and/or variable regions, and wherein the endogenousimmunoglobulin encoding loci are substituted or inactivated. WO 96/30498and U.S. Pat. No. 6,091,001 disclose the use of the Cre/Lox system tomodify the immunoglobulin locus in a mammal, such as to replace all or aportion of the constant or variable region to form a modified antibodymolecule. WO 94/02602 discloses non-human mammalian hosts havinginactivated endogenous Ig loci and functional human Ig loci. U.S. Pat.No. 5,939,598 discloses methods of making transgenic mice in which themice lack endogenous heavy chains, and express an exogenousimmunoglobulin locus comprising one or more xenogeneic constant regions.See also, U.S. Pat. Nos. 6,114,598 6,657,103 and 6,833,268.

Using a transgenic animal described above, an immune response can beproduced to a selected antigen, and antibody producing cells can beremoved from the animal and used to produce hybridomas that secretehuman monoclonal antibodies Immunization protocols, adjuvants, and thelike are known in the art, and are used in immunization of, for example,a transgenic mouse as described in WO 96/33735. This publicationdiscloses monoclonal antibodies against a variety of antigens includingIL-6, IL-8, TNFa, human CD4, L selectin, gp39, and tetanus toxin. Themonoclonal antibodies can be tested for the ability to inhibit orneutralize the biological activity or physiological effect of thecorresponding protein. WO 96/33735 discloses that monoclonal antibodiesagainst IL-8, derived from immune cells of transgenic mice immunizedwith IL-8, blocked IL-8 induced functions of neutrophils. Humanmonoclonal antibodies with specificity for the antigen used to immunizetransgenic animals are also disclosed in WO 96/34096 and U.S. patentapplication no. 20030194404; and U.S. patent application no.20030031667.

Additional transgenic animals useful to make monoclonal antibodiesinclude the Medarex HuMAb-MOUSE®, described in U.S. Pat. No. 5,770,429and Fishwild, et al. (Nat. Biotechnol. 14:845-851 (1996)), whichcontains gene sequences from unrearranged human antibody genes that codefor the heavy and light chains of human antibodies Immunization of aHuMAb-MOUSE® enables the production of fully human monoclonal antibodiesto the antigen.

Also, Ishida et al. (Cloning Stem Cells. 4:91-102 (2002)) describes theTransChromo Mouse (TCMOUSE™) which comprises megabase-sized segments ofhuman DNA and which incorporates the entire human immunoglobulin (hIg)loci. The TCMOUSE™ has a fully diverse repertoire of hIgs, including allthe subclasses of IgGs (IgG1-G4) Immunization of the TCMOUSE™ withvarious human antigens produces antibody responses comprising humanantibodies.

See also Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993);Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Yearin Immunol., 7:33 (1993); and U.S. Pat. No. 5,591,669, U.S. Pat. No.5,589,369, U.S. Pat. No. 5,545,807; and U.S. Patent Publication No.20020199213. U.S. Patent Publication No. 20030092125 describes methodsfor biasing the immune response of an animal to the desired epitope.Human antibodies may also be generated by in vitro activated B cells(see U.S. Pat. Nos. 5,567,610 and 5,229,275).

Human Antibodies from Display Technology

The development of technologies for making repertoires of recombinanthuman antibody genes, and the display of the encoded antibody fragmentson the surface of filamentous bacteriophage, has provided a means formaking human antibodies directly. The antibodies produced by phagetechnology are produced as antigen binding fragments-usually Fv or Fabfragments-in bacteria and thus lack effector functions. Effectorfunctions can be introduced by one of two strategies: The fragments canbe engineered either into complete antibodies for expression inmammalian cells, or into bispecific antibody fragments with a secondbinding site capable of triggering an effector function.

The invention contemplates a method for producing antigen-specificantibody or antigen-binding portion thereof comprising the steps ofsynthesizing a library of human antibodies on phage, screening thelibrary with antigen or a portion thereof, isolating phage that bindantigen, and obtaining the antibody from the phage. By way of example,one method for preparing the library of antibodies for use in phagedisplay techniques comprises the steps of immunizing a non-human animalcomprising human immunoglobulin loci with antigen or an antigenicportion thereof to create an immune response, extracting antibodyproducing cells from the immunized animal; isolating RNA from theextracted cells, reverse transcribing the RNA to produce cDNA,amplifying the cDNA using a primer, and inserting the cDNA into a phagedisplay vector such that antibodies are expressed on the phage.Recombinant antigen-specific antibodies of the invention may be obtainedin this way. In another example, antibody producing cells can beextracted from non-immunized animals, RNA isolated from the extractedcells and reverse transcribed to produce cDNA, which is amplified usinga primer, and inserted into a phage display vector such that antibodiesare expressed on the phage. Phage-display processes mimic immuneselection through the display of antibody repertoires on the surface offilamentous bacteriophage, and subsequent selection of phage by theirbinding to an antigen of choice. One such technique is described in WO99/10494, which describes the isolation of high affinity and functionalagonistic antibodies for MPL and msk receptors using such an approach.Antibodies of the invention can be isolated by screening of arecombinant combinatorial antibody library, preferably a scFv phagedisplay library, prepared using human V_(L) and V_(H) cDNAs preparedfrom mRNA derived from human lymphocytes. Methodologies for preparingand screening such libraries are known in the art. See e.g., U.S. Pat.No. 5,969,108. There are commercially available kits for generatingphage display libraries (e.g., the Pharmacia Recombinant Phage AntibodySystem, catalog no. 27-9400-01; and the Stratagene SurfZAP™ phagedisplay kit, catalog no. 240612). There are also other methods andreagents that can be used in generating and screening antibody displaylibraries (see, e.g., Ladner et al. U.S. Pat. No. 5,223,409; Kang et al.PCT Publication No. WO 92/18619; Dower et al. PCT Publication No. WO91/17271; Winter et al. PCT Publication No. WO 92/20791; Markland et al.PCT Publication No. WO 92/15679; Breitling et al. PCT Publication No. WO93/01288; McCafferty et al. PCT Publication No. WO 92/01047; Garrard etal. PCT Publication No. WO 92/09690; Fuchs et al. (1991) Bio/Technology9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse etal. (1989) Science 246:1275-1281; McCafferty et al., Nature (1990)348:552-554; Griffiths et al. (1993) EMBO J 12:725-734; Hawkins et al.(1992) J. Mol. Biol. 226:889-896; Clackson et al. (1991) Nature352:624-628; Gram et al. (1992) Proc. Natl. Acad. Sci. USA 89:3576-3580;Garrad et al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al.(1991) Nuc Acid Res 19:4133-4137; and Barbas et al. (1991) Proc. Natl.Acad. Sci. USA 88:7978-7982.

In one embodiment, to isolate human antibodies specific for an antigen,with the desired binding characteristics, a human V_(H) and V_(L)library are screened to select for antibody fragments having the desiredspecificity. The antibody libraries used in this method are preferablyscFv libraries prepared and screened as described herein and in the art(McCafferty et al., PCT Publication No. WO 92/01047, McCafferty et al.,(Nature 348:552-554 (1990)); and Griffiths et al., (EMBO J 12:725-734(1993)). The scFv antibody libraries preferably are screened using theantigen.

Alternatively, the Fd fragment (V_(H)-C_(H)1) and light chain(V_(L)-C_(L)) of antibodies are separately cloned by PCR and recombinedrandomly in combinatorial phage display libraries, which can then beselected for binding to a particular antigen. The Fab fragments areexpressed on the phage surface, i.e., physically linked to the genesthat encode them. Thus, selection of Fab by antigen binding co-selectsfor the Fab encoding sequences, which can be amplified subsequently.Through several rounds of antigen binding and re-amplification, aprocedure termed panning, Fab specific for the antigen are enriched andfinally isolated.

In 1994, an approach for the humanization of antibodies, called “guidedselection”, was described. Guided selection utilizes the power of thephage display technique for the humanization of mouse monoclonalantibody (See Jespers, L. S., et al., Bio/Technology 12, 899-903(1994)). For this, the Fd fragment of the mouse monoclonal antibody canbe displayed in combination with a human light chain library, and theresulting hybrid Fab library may then be selected with antigen. Themouse Fd fragment thereby provides a template to guide the selection.Subsequently, the selected human light chains are combined with a humanFd fragment library. Selection of the resulting library yields entirelyhuman Fab.

A variety of procedures have been described for deriving humanantibodies from phage-display libraries (See, for example, Hoogenboom etal., J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol,222:581-597 (1991); U.S. Pat. Nos. 5,565,332 and 5,573,905; Clackson,T., and Wells, J. A., TIBTECH 12, 173-184 (1994)). In particular, invitro selection and evolution of antibodies derived from phage displaylibraries has become a powerful tool (See Burton, D. R., and Barbas III,C. F., Adv. Immunol. 57, 191-280 (1994); Winter, G., et al., Annu. Rev.Immunol. 12, 433-455 (1994); U.S. patent publication no. 20020004215 andWO 92/01047; U.S. patent publication no. 20030190317; and U.S. Pat. Nos.6,054,287 and 5,877,293.

Watkins, “Screening of Phage-Expressed Antibody Libraries by CaptureLift,” Methods in Molecular Biology, Antibody Phage Display: Methods andProtocols 178:187-193 (2002), and U.S. patent publication no.20030044772, published Mar. 6, 2003, describe methods for screeningphage-expressed antibody libraries or other binding molecules by capturelift, a method involving immobilization of the candidate bindingmolecules on a solid support.

Fv fragments are displayed on the surface of phage, by the associationof one chain expressed as a phage protein fusion (e.g., with M13 geneIII) with the complementary chain expressed as a soluble fragment. It iscontemplated that the phage may be a filamentous phage such as one ofthe class I phages: fd, M13, fl, Ifl, lke, ZJ/Z, Ff and one of the classII phages Xf, Pf1 and Pf3. The phage may be M13, or fd or a derivativethereof.

Once initial human V_(L) and V_(H) segments are selected, “mix andmatch” experiments, in which different pairs of the initially selectedV_(L) and V_(H) segments are screened for antigen binding, may beperformed to select preferred V_(L)/V_(H) pair combinations.Additionally, to further improve the quality of the antibody, the V_(L)and V_(H) segments of the preferred V_(L)/V_(H) pair(s) can be randomlymutated, preferably within the any of the CDR1, CDR2 or CDR3 region ofV_(H) and/or V_(L), in a process analogous to the in vivo somaticmutation process responsible for affinity maturation of antibodiesduring a natural immune response. This in vitro affinity maturation canbe accomplished by amplifying V_(L) and V_(H) regions using PCR primerscomplimentary to the V_(H) CDR1, CDR2, and CDR3, or V_(L) CDR1, CDR2,and CDR3, respectively, which primers have been “spiked” with a randommixture of the four nucleotide bases at certain positions such that theresultant PCR products encode V_(L) and V_(H) segments into which randommutations have been introduced into the V_(H) and/or V_(L) CDR3 regions.These randomly mutated V_(L) and V_(H) segments can be rescreened forbinding to antigen.

Following screening and isolation of an antigen-specific antibody from arecombinant immunoglobulin display library, nucleic acid encoding theselected antibody can be recovered from the display package (e.g., fromthe phage genome) and subcloned into other expression vectors bystandard recombinant DNA techniques. If desired, the nucleic acid can befurther manipulated to create other antibody forms of the invention, asdescribed below. To express a recombinant human antibody isolated byscreening of a combinatorial library, the DNA encoding the antibody iscloned into a recombinant expression vector and introduced into amammalian host cell, as described herein.

It is contemplated that the phage display method may be carried out in amutator strain of bacteria or host cell. A mutator strain is a host cellwhich has a genetic defect which causes DNA replicated within it to bemutated with respect to its parent DNA. Example mutator strains areNR9046mutD5 and NR9046 mut T1.

It is also contemplated that the phage display method may be carried outusing a helper phage. This is a phage which is used to infect cellscontaining a defective phage genome and which functions to complementthe defect. The defective phage genome can be a phagemid or a phage withsome function encoding gene sequences removed. Examples of helper phagesare M13K07, M13K07 gene III no. 3, hyperphage; and phage displaying orencoding a binding molecule fused to a capsid protein.

Antibodies may also be generated via phage display screening methodsusing the hierarchical dual combinatorial approach as disclosed in WO92/01047 in which an individual colony containing either an H or L chainclone is used to infect a complete library of clones encoding the otherchain (L or H) and the resulting two-chain specific binding member isselected in accordance with phage display techniques such as thosedescribed therein. This technique is also disclosed in Marks et al,(Bio/Technology, 10:779-783 (1992)).

Methods for display of polypeptides on the surface of viruses, yeast,microbial and mammalian cells have also been used to identify antigenspecific antibodies. See, for example, U.S. Pat. Nos. 5,348,867;5,723,287; 6,699,658; Wittrup, Curr Op. Biotech. 12:395-99 (2001); Leeet al, Trends in Biotech. 21(1) 45-52 (2003); Surgeeva et al, Adv. DrugDeliv. Rev. 58: 1622-54 (2006). Antibody libraries may be attached toyeast proteins, such as agglutinin, effectively mimicking the cellsurface display of antibodies by B cells in the immune system.

In addition to phage display methods, antibodies may be isolated usingin vitro display methods including ribosome display and mRNA display(Amstutz et al, Curr. Op. Biotech. 12: 400-05 (2001)). Selection ofpolypeptide using ribosome display is described in Hanes et al., (Proc.Natl Acad Sci USA, 94:4937-4942 (1997)) and U.S. Pat. Nos. 5,643,768 and5,658,754 issued to Kawasaki. Ribosome display is also useful for rapidlarge scale mutational analysis of antibodies. The selective mutagenesisapproach also provides a method of producing antibodies with improvedactivities that can be selected using ribosomal display techniques.

Altered Glycosylation

Antibody variants can also be produced that have a modifiedglycosylation pattern relative to the parent antibody, for example,deleting one or more carbohydrate moieties found in the antibody, and/oradding one or more glycosylation sites that are not present in theantibody.

Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain. Thepresence of either of these tripeptide sequences in a polypeptidecreates a potential glycosylation site. Thus, N-linked glycosylationsites may be added to an antibody by altering the amino acid sequencesuch that it contains one or more of these tripeptide sequences.O-linked glycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used. O-linked glycosylation sites may beadded to an antibody by inserting or substituting one or more serine orthreonine residues to the sequence of the original antibody.

Fc glycans influence the binding of IgG to Fc receptors and C1q, and aretherefore important for IgG effector functions. Antibody variants withmodified Fc glycans and altered effector function may be produced. Forexample, antibodies with modified terminal sugars such as sialic acids,core fucose, bisecting N-acetylglucosamine, and mannose residues mayhave altered binding to the FcγRIIIa receptor and altered ADCC activity.In a further example, antibodies with modified terminal galactoseresidues may have altered binding to C1q and altered CDC activity (Raju,Curr. Opin. Immunol. 20: 471-78 (2008).

Also contemplated are antibody molecules with absent or reducedfucosylation that exhibit improved ADCC activity. A variety of ways areknown in the art to accomplish this. For example, ADCC effector activityis mediated by binding of the antibody molecule to the FcγRIII receptor,which has been shown to be dependent on the carbohydrate structure ofthe N-linked glycosylation at the Asn-297 of the CH2 domain.Non-fucosylated antibodies bind this receptor with increased affinityand trigger FcγRIII-mediated effector functions more efficiently thannative, fucosylated antibodies. For example, recombinant production ofnon-fucosylated antibody in CHO cells in which the alpha-1,6-fucosyltransferase enzyme has been knocked out results in antibody with100-fold increased ADCC activity (Yamane-Ohnuki et al., BiotechnolBioeng. 87:614-22 (2004)). Similar effects can be accomplished throughdecreasing the activity of this or other enzymes in the fucosylationpathway, e.g., through siRNA or antisense RNA treatment, engineeringcell lines to knockout the enzyme(s), or culturing with selectiveglycosylation inhibitors (Rothman et al., Mol Immunol. 26:1113-23(1989)). Some host cell strains, e.g. Lec13 or rat hybridoma YB2/0 cellline naturally produce antibodies with lower fucosylation levels.(Shields et al., J Biol Chem. 277:26733-40 (2002); Shinkawa et al., JBiol Chem. 278:3466-73 (2003)). An increase in the level of bisectedcarbohydrate, e.g. through recombinantly producing antibody in cellsthat overexpress GnTIII enzyme, has also been determined to increaseADCC activity (Umana et al., Nat Biotechnol. 17:176-80 (1999)). It hasbeen predicted that the absence of only one of the two fucose residuesmay be sufficient to increase ADCC activity (Ferrara et al., BiotechnolBioeng. 93:851-61 (2006)).

Variants with Altered Effector Function

Other modifications of the antibody are contemplated. In one aspect, itmay be desirable to modify the antibody of the invention with respect toeffector function, for example, to enhance the effectiveness of theantibody in treating cancer (Natsume et al, Drug Design Dev't & Ther. 3:7-16 (2009). Exemplary effector functions include C1q binding; CDC; Fcreceptor binding; ADCC; phagocytosis; down regulation of cell surfacereceptors (e.g. B cell receptor; BCR), etc. One method for modifyingeffector function teaches that cysteine residue(s) may be introduced inthe Fc region, thereby allowing interchain disulfide bond formation inthis region. The homodimeric antibody thus generated may have improvedinternalization capability and/or increased complement-mediated cellkilling and antibody-dependent cellular cytotoxicity (ADCC). See Caronet al., (J. Exp Med. 176: 1191-1195 (1992)) and Shopes, B. (J. Immunol.148: 2918-2922 (1992)). Homodimeric antibodies with enhanced anti-tumoractivity may also be prepared using heterobifunctional cross-linkers asdescribed in Wolff et al., (Cancer Research 53: 2560-2565 (1993)).Alternatively, an antibody can be engineered which has dual Fc regionsand may thereby have enhanced complement lysis and ADCC capabilities.See Stevenson et al., (Anti-Cancer Drug Design 3: 219-230 (1989)). Inaddition, it has been shown that sequences within the CDR can cause anantibody to bind to MHC Class II and trigger an unwanted helper T-cellresponse. A conservative substitution can allow the antibody to retainbinding activity yet lose its ability to trigger an unwanted T-cellresponse. Also see Steplewski et al., (Proc Natl Acad Sci USA.85:4852-56 (1998)), which described chimeric antibodies wherein a murinevariable region was joined with human gamma 1, gamma 2, gamma 3, andgamma 4 constant regions.

In certain embodiments of the invention, it may be desirable to use anantibody fragment, rather than an intact antibody, to increase tumorpenetration, for example. In this case, it may be desirable to modifythe antibody fragment in order to increase its serum half-life, forexample, adding molecules such as PEG or other water soluble polymers,including polysaccharide polymers, to antibody fragments to increase thehalf-life. This may also be achieved, for example, by incorporation of asalvage receptor binding epitope into the antibody fragment (e.g., bymutation of the appropriate region in the antibody fragment or byincorporating the epitope into a peptide tag that is then fused to theantibody fragment at either end or in the middle, e.g., by DNA orpeptide synthesis) (see, e.g., WO96/32478).

The salvage receptor binding epitope preferably constitutes a regionwherein any one or more amino acid residues from one or two loops of anFc domain are transferred to an analogous position of the antibodyfragment. Even more preferably, three or more residues from one or twoloops of the Fc domain are transferred. Still more preferred, theepitope is taken from the CH2 domain of the Fc region (e.g., of an IgG)and transferred to the CH1, CH3, or VH region, or more than one suchregion, of the antibody. Alternatively, the epitope is taken from theCH2 domain of the Fc region and transferred to the C_(L) region or V_(L)region, or both, of the antibody fragment. See also Internationalapplications WO 97/34631 and WO 96/32478 which describe Fc variants andtheir interaction with the salvage receptor.

Thus, antibodies of the invention may comprise a human Fc portion, ahuman consensus Fc portion, or a variant thereof that retains theability to interact with the Fc salvage receptor, including variants inwhich cysteines involved in disulfide bonding are modified or removed,and/or in which the a met is added at the N-terminus and/or one or moreof the N-terminal 20 amino acids are removed, and/or regions thatinteract with complement, such as the C1q binding site, are removed,and/or the ADCC site is removed [see, e.g., Sarmay et al., Molec.Immunol. 29:633-9 (1992)].

Previous studies mapped the binding site on human and murine IgG for FcRprimarily to the lower hinge region composed of IgG residues 233-239.Other studies proposed additional broad segments, e.g. G1γ316-Lys338 forhuman Fc receptor I, Lys274-Arg301 and Tyr407-Arg416 for human Fcreceptor III, or found a few specific residues outside the lower hinge,e.g., Asn297 and Glu318 for murine IgG2b interacting with murine Fcreceptor II. The report of the 3.2-Å crystal structure of the human IgG1Fc fragment with human Fc receptor IIIA delineated IgG1 residuesLeu234-Ser239, Asp265-Glu269, Asn297-Thr299, and Ala327-Ile332 asinvolved in binding to Fc receptor IIIA. It has been suggested based oncrystal structure that in addition to the lower hinge (Leu234-G1γ237),residues in IgG CH2 domain loops FG (residues 326-330) and BC (residues265-271) might play a role in binding to Fc receptor IIA. See Shields etal., (J. Biol. Chem., 276:6591-604 (2001)), incorporated by referenceherein in its entirety. Mutation of residues within Fc receptor bindingsites can result in altered effector function, such as altered ADCC orCDC activity, or altered half-life. As described above, potentialmutations include insertion, deletion or substitution of one or moreresidues, including substitution with alanine, a conservativesubstitution, a non-conservative substitution, or replacement with acorresponding amino acid residue at the same position from a differentIgG subclass (e.g. replacing an IgG1 residue with a corresponding IgG2residue at that position).

Shields et al. reported that IgG1 residues involved in binding to allhuman Fc receptors are located in the CH2 domain proximal to the hingeand fall into two categories as follows: 1) positions that may interactdirectly with all FcR include Leu234-Pro238, Ala327, and Pro329 (andpossibly Asp265); 2) positions that influence carbohydrate nature orposition include Asp265 and Asn297. The additional IgG1 residues thataffected binding to Fc receptor II are as follows: (largest effect)Arg255, Thr256, Glu258, Ser267, Asp270, Glu272, Asp280, Arg292, Ser298,and (less effect) His268, Asn276, His285, Asn286, Lys290, Gln295,Arg301, Thr307, Leu309, Asn315, Lys322, Lys326, Pro331, Ser337, Ala339,Ala378, and Lys414. A327Q, A327S, P329A, D265A and D270A reducedbinding. In addition to the residues identified above for all FcR,additional IgG1 residues that reduced binding to Fc receptor IIIA by 40%or more are as follows: Ser239, Ser267 (Gly only), His268, Glu293,Gln295, Tyr296, Arg301, Val303, Lys338, and Asp376. Variants thatimproved binding to FcRIIIA include T256A, K290A, S298A, E333A, K334A,and A339T. Lys414 showed a 40% reduction in binding for FcRIIA andFcRIIB, Arg416 a 30% reduction for FcRIIA and FcRIIIA, Gln419 a 30%reduction to FcRIIA and a 40% reduction to FcRIIB, and Lys360 a 23%improvement to FcRIIIA See also Presta et al., (Biochem. Soc. Trans.30:487-490, 2001), incorporated herein by reference in its entirety,which described several positions in the Fc region of IgG1 were foundwhich improved binding only to specific Fc gamma receptors (R) orsimultaneously improved binding to one type of Fc gamma R and reducedbinding to another type. Selected IgG1 variants with improved binding toFc gamma RIIIa were then tested in an in vitro antibody-dependentcellular cytotoxicity (ADCC) assay and showed an enhancement in ADCCwhen either peripheral blood mononuclear cells or natural killer cellswere used.

For example, U.S. Pat. No. 6,194,551, incorporated herein by referencein its entirety, describes variants with altered effector functioncontaining mutations in the human IgG Fc region, at amino acid position329, 331 or 322 (using Kabat numbering), some of which display reducedC1q binding or CDC activity. As another example, U.S. Pat. No.6,737,056, incorporated herein by reference in its entirety, describesvariants with altered effector or Fc-gamma-receptor binding containingmutations in the human IgG Fc region, at amino acid position 238, 239,248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276,278, 280, 283, 285, 286, 289, 290, 292, 294, 295, 296, 298, 301, 303,305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333,334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414,416, 419, 430, 434, 435, 437, 438 or 439 (using Kabat numbering), someof which display receptor binding profiles associated with reduced ADCCor CDC activity. Of these, a mutation at amino acid position 238, 265,269, 270, 327 or 329 are stated to reduce binding to FcRI, a mutation atamino acid position 238, 265, 269, 270, 292, 294, 295, 298, 303, 324,327, 329, 333, 335, 338, 373, 376, 414, 416, 419, 435, 438 or 439 arestated to reduce binding to FcRII, and a mutation at amino acid position238, 239, 248, 249, 252, 254, 265, 268, 269, 270, 272, 278, 289, 293,294, 295, 296, 301, 303, 322, 327, 329, 338, 340, 373, 376, 382, 388,389, 416, 434, 435 or 437 is stated to reduce binding to FcRIII.

U.S. Pat. No. 5,624,821, incorporated by reference herein in itsentirety, reports that C1q binding activity of an murine antibody can bealtered by mutating amino acid residue 318, 320 or 322 of the heavychain and that replacing residue 297 (Asn) results in removal of lyticactivity.

U.S. Patent Publication No. 20040132101, incorporated by referenceherein in its entirety, describes variants with mutations at amino acidpositions 240, 244, 245, 247, 262, 263, 266, 299, 313, 325, 328, or 332(using Kabat numbering) or positions 234, 235, 239, 240, 241, 243, 244,245, 247, 262, 263, 264, 265, 266, 267, 269, 296, 297, 298, 299, 313,325, 327, 328, 329, 330, or 332 (using Kabat numbering), of whichmutations at positions 234, 235, 239, 240, 241, 243, 244, 245, 247, 262,263, 264, 265, 266, 267, 269, 296, 297, 298, 299, 313, 325, 327, 328,329, 330, or 332 may reduce ADCC activity or reduce binding to an Fcgamma receptor.

Chappel et al. (Proc Natl Acad Sci USA. 88:9036-40 (1991)), incorporatedherein by reference in its entirety, report that cytophilic activity ofIgG1 is an intrinsic property of its heavy chain CH2 domain. Singlepoint mutations at any of amino acid residues 234-237 of IgG1significantly lowered or abolished its activity. Substitution of all ofIgG1 residues 234-237 (LLGG) into IgG2 and IgG4 were required to restorefull binding activity. An IgG2 antibody containing the entire ELLGGPsequence (residues 233-238) was observed to be more active thanwild-type IgG1.

Isaacs et al. (J Immunol. 161:3862-9 (1998)), incorporated herein byreference in its entirety, report that mutations within a motif criticalfor Fc gammaR binding (glutamate 233 to proline, leucine/phenylalanine234 to valine, and leucine 235 to alanine) completely preventeddepletion of target cells. The mutation glutamate 318 to alanineeliminated effector function of mouse IgG2b and also reduced the potencyof human IgG4.

Armour et al. (Mol Immunol. 40:585-93 (2003)), incorporated by referenceherein in its entirety, identified IgG1 variants which react with theactivating receptor, FcgammaRIIa, at least 10-fold less efficiently thanwildtype IgG1 but whose binding to the inhibitory receptor, FcgammaRIIb,is only four-fold reduced. Mutations were made in the region of aminoacids 233-236 and/or at amino acid positions 327, 330 and 331. See alsoWO 99/58572, incorporated by reference herein in its entirety.

Xu et al. (J Biol Chem. 269:3469-74 (1994)), incorporated by referenceherein in its entirety, report that mutating IgG1 Pro331 to Ser markedlydecreased C1q binding and virtually eliminated lytic activity. Incontrast, the substitution of Pro for Ser331 in IgG4 bestowed partiallytic activity (40%) to the IgG4 Pro331 variant.

Schuurman et al. (Mol Immunol. 38:1-8 (2001)), incorporated by referenceherein in its entirety, report that mutating one of the hinge cysteinesinvolved in the inter-heavy chain bond formation, Cys226, to serineresulted in a more stable inter-heavy chain linkage. Mutating the IgG4hinge sequence Cys-Pro-Ser-Cys to the IgG1 hinge sequenceCys-Pro-Pro-Cys also markedly stabilizes the covalent interactionbetween the heavy chains.

Angal et al. (Mol Immunol. 30:105-8 (1993)), incorporated by referenceherein in its entirety, report that mutating the serine at amino acidposition 241 in IgG4 to proline (found at that position in IgG1 andIgG2) led to the production of a homogeneous antibody, as well asextending serum half-life and improving tissue distribution compared tothe original chimeric IgG4.

Covalent Modifications

Covalent modifications of the polypeptide binding agents of theinvention, e.g., antibodies, are also included within the scope of thisinvention. They may be made by chemical synthesis or by enzymatic orchemical cleavage of the polypeptide binding agent, if applicable. Othertypes of covalent modifications of the polypeptide binding agent areintroduced into the molecule by reacting targeted amino acid residues ofthe polypeptide binding agent with an organic derivatizing agent that iscapable of reacting with selected side chains or the N- or C-terminalresidues.

Cysteinyl residues most commonly are reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesalso are derivatized by reaction with bromotrifluoroacetone,.alpha.-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1 M sodium cacodylate at pH 6.0.

Lysinyl and amino-terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing .alpha-amino-containing residues includeimidoesters such as methyl picolinimidate, pyridoxal phosphate,pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid,O-methylisourea, 2,4-pentanedione, and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pK_(a) of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane are used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosylresidues are iodinated using ¹²⁵I or ¹³¹I to prepare labeled proteinsfor use in radioimmunoassay.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R—N═C═N—R′), where R and R′ are differentalkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.Furthermore, aspartyl and glutamyl residues are converted to asparaginyland glutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues, respectively. Theseresidues are deamidated under neutral or basic conditions. Thedeamidated form of these residues falls within the scope of thisinvention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MolecularProperties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)),acetylation of the N-terminal amine, and amidation of any C-terminalcarboxyl group.

Another type of covalent modification involves chemically orenzymatically coupling glycosides to the polypeptide binding agent.These procedures are advantageous in that they do not require productionof the polypeptide binding agent in a host cell that has glycosylationcapabilities for N- or O-linked glycosylation. Depending on the couplingmode used, the sugar(s) may be attached to (a) arginine and histidine,(b) free carboxyl groups, (c) free sulfhydryl groups such as those ofcysteine, (d) free hydroxyl groups such as those of serine, threonine,or hydroxyproline, (e) aromatic residues such as those of phenylalanine,tyrosine, or tryptophan, or (f) the amide group of glutamine. Thesemethods are described in WO87/05330 and in Aplin and Wriston, (CRC Crit.Rev. Biochem., pp. 259-306 (1981)).

Removal of any carbohydrate moieties present on the polypeptide bindingagent may be accomplished chemically or enzymatically. Chemicaldeglycosylation requires exposure of the polypeptide binding agent tothe compound trifluoromethanesulfonic acid, or an equivalent compound.This treatment results in the cleavage of most or all sugars except thelinking sugar (N-acetylglucosamine or N-acetylgalactosamine), whileleaving the polypeptide binding agent intact. Chemical deglycosylationis described by Hakimuddin, et al., (Arch. Biochem. Biophys. 259: 52(1987)) and by Edge et al., (Anal. Biochem. 118: 131 (1981)). Enzymaticcleavage of carbohydrate moieties on polypeptide binding agents can beachieved by the use of a variety of endo- and exo-glycosidases asdescribed by Thotakura et al., (Meth. Enzymol. 138: 350 (1987)).

Another type of covalent modification of the polypeptide binding agentcomprises linking the polypeptide binding agent to one of a variety ofhydrophobic moieties or nonproteinaceous polymers, e.g., polyethyleneglycol, polypropylene glycol, polyoxyethylated polyols, polyoxyethylatedsorbitol, polyoxyethylated glucose, polyoxyethylated glycerol,polyoxyalkylenes, or polysaccharide polymers such as dextran. Suchmethods are known in the art, see, e.g. U.S. Pat. Nos. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192, 4,179,337, 4,766,106,4,179,337, 4,495,285, 4,609,546 or EP 315 456.

Derivatives

Derivative refers to polypeptide binding agents, including antibodies,chemically modified by such techniques as ubiquitination, labeling(e.g., with radionuclides or various enzymes), covalent polymerattachment such as pegylation (derivatization with polyethylene glycol)and insertion or substitution by chemical synthesis of amino acids suchas ornithine. Derivatives of the polypeptide binding agents of theinvention, such as an antibody, are also useful as therapeutic agentsand may be produced by the method of the invention

The conjugated moiety can be incorporated in or attached to apolypeptide binding agent either covalently, or through ionic, van derWaals or hydrogen bonds, e.g., incorporation of radioactive nucleotides,or biotinylated nucleotides that are recognized by streptavadin.

Polyethylene glycol (PEG) may be attached to the polypeptide bindingagents to provide a longer half-life in vivo. The PEG group may be ofany convenient molecular weight and may be linear or branched. Theaverage molecular weight of the PEG will preferably range from about 2kiloDalton (“kD”) to about 100 kDa, more preferably from about 5 kDa toabout 50 kDa, most preferably from about 5 kDa to about 10 kDa. The PEGgroups will generally be attached to the polypeptide binding agents ofthe invention via acylation or reductive alkylation through a natural orengineered reactive group on the PEG moiety (e.g., an aldehyde, amino,thiol, or ester group) to a reactive group on the polypeptide bindingagent (e.g., an aldehyde, amino, or ester group). Addition of PEGmoieties to polypeptide binding agents can be carried out usingtechniques well-known in the art. See, e.g., International PublicationNo. WO 96/11953 and U.S. Pat. No. 4,179,337.

Ligation of the polypeptide binding agent with PEG usually takes placein aqueous phase and can be easily monitored by reverse phase analyticalHPLC. The PEGylated substances are purified by preparative HPLC andcharacterized by analytical HPLC, amino acid analysis and laserdesorption mass spectrometry.

Antibody Conjugates

A polypeptide binding agent may be administered in its “naked” orunconjugated form, or may be conjugated directly to other therapeutic ordiagnostic agents, or may be conjugated indirectly to carrier polymerscomprising such other therapeutic or diagnostic agents. In someembodiments the polypeptide binding agent is conjugated to a cytotoxicagent such as a chemotherapeutic agent, a drug, a growth inhibitoryagent, a toxin (e.g., an enzymatically active toxin of bacterial,fungal, plant, or animal origin, or fragments thereof), or a radioactiveisotope (i.e., a radioconjugate). Suitable chemotherapeutic agentsinclude: daunomycin, doxorubicin, methotrexate, and vindesine (Rowlandet al., (1986) supra). Suitable toxins include: bacterial toxins such asdiphtheria toxin; plant toxins such as ricin; small molecule toxins suchas geldanamycin (Mandler et al J. Natl. Cancer Inst. 92(19):1573-81(2000); Mandler et al., Bioorg. Med. Chem. Letters 10:1025-1028 (2000);Mandler et al., Bioconjugate Chem. 13.786-91 (2002)), maytansinoids (EP1391213; Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-23 (1996)),auristatins (Doronina et al., Nat. Biotech. 21: 778-84 (2003) andcalicheamicin (Lode et al., Cancer Res. 58:2928 (1998); Hinman et al.,Cancer Res. 53:3336-3342 (1993)).

Polypeptide binding agents can be detectably labeled through the use ofradioisotopes, affinity labels (such as biotin, avidin, etc.), enzymaticlabels (such as horseradish peroxidase, alkaline phosphatase, etc.)fluorescent or luminescent or bioluminescent labels (such as FITC orrhodamine, etc.), paramagnetic atoms, and the like. Procedures foraccomplishing such labeling are well known in the art; for example, see(Sternberger, L. A. et al., J. Histochem. Cytochem. 18:315 (1970);Bayer, E. A. et al., Meth. Enzym. 62:308 (1979); Engval, E. et al.,Immunol. 109:129 (1972); Goding, J. W. J. Immunol. Meth. 13:215 (1976)).

Conjugation of polypeptide binding agent moieties is described in U.S.Pat. No. 6,306,393. General techniques are also described in Shih etal., Int. J. Cancer 41:832-839 (1988); Shih et al., Int. J. Cancer46:1101-1106 (1990); and Shih et al., U.S. Pat. No. 5,057,313. Thisgeneral method involves reacting a polypeptide binding agent componenthaving an oxidized carbohydrate portion with a carrier polymer that hasat least one free amine function and that is loaded with a plurality ofdrug, toxin, chelator, boron addends, or other therapeutic agent. Thisreaction results in an initial Schiff base (imine) linkage, which can bestabilized by reduction to a secondary amine to form the finalconjugate.

The carrier polymer may be, for example, an aminodextran or polypeptideof at least 50 amino acid residues. Various techniques for conjugating adrug or other agent to the carrier polymer are known in the art. Apolypeptide carrier can be used instead of aminodextran, but thepolypeptide carrier should have at least 50 amino acid residues in thechain, preferably 100-5000 amino acid residues. At least some of theamino acids should be lysine residues or glutamate or aspartateresidues. The pendant amines of lysine residues and pendant carboxylatesof glutamine and aspartate are convenient for attaching a drug, toxin,immunomodulator, chelator, boron addend or other therapeutic agent.Examples of suitable polypeptide carriers include polylysine,polyglutamic acid, polyaspartic acid, co-polymers thereof, and mixedpolymers of these amino acids and others, e.g., serines, to conferdesirable solubility properties on the resultant loaded carrier andconjugate.

Alternatively, conjugated polypeptide binding agents can be prepared bydirectly conjugating a polypeptide binding agent component with atherapeutic agent. The general procedure is analogous to the indirectmethod of conjugation except that a therapeutic agent is directlyattached to an oxidized polypeptide binding agent component. Forexample, a carbohydrate moiety of a polypeptide binding agent can beattached to polyethyleneglycol to extend half-life.

Alternatively, a therapeutic agent can be attached at the hinge regionof a reduced antibody component via disulfide bond formation, or using aheterobifunctional cross-linker, such as N-succinyl3-(2-pyridyldithio)proprionate (SPDP). Yu et al., Int. J. Cancer 56:244(1994). General techniques for such conjugation are well-known in theart. See, for example, Wong, Chemistry Of Protein Conjugation andCross-Linking (CRC Press 1991); Upeslacis et al., “Modification ofAntibodies by Chemical Methods,” in Monoclonal Antibodies: Principlesand Applications, Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc.1995); Price, “Production and Characterization of SyntheticPeptide-Derived Antibodies,” in Monoclonal Antibodies: Production,Engineering and Clinical Application, Ritter et al. (eds.), pages 60-84(Cambridge University Press 1995). A variety of bifunctional proteincoupling agents are known in the art, such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such asbis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene).

Antibody Fusion Proteins

Methods of making antibody fusion proteins are well known in the art.See, e.g., U.S. Pat. No. 6,306,393. Antibody fusion proteins comprisingan interleukin-2 moiety are described by Boleti et al., Ann. Oncol.6:945 (1995), Nicolet et al., Cancer Gene Ther. 2:161 (1995), Becker etal., Proc. Nat'l Acad. Sci. USA 93:7826 (1996), Hank et al., Clin.Cancer Res. 2:1951 (1996), and Hu et al., Cancer Res. 56:4998 (1996). Inaddition, Yang et al., (Hum. Antibodies Hybridomas 6:129 (1995)),describe a fusion protein that includes an F(ab′)₂ fragment and a tumornecrosis factor alpha moiety. Further examples of antibody fusionproteins are described by Pastan et al, Nat. Reviews Cancer 6: 559-65(2006).

Methods of making antibody-toxin fusion proteins in which a recombinantmolecule comprises one or more antibody components and a toxin orchemotherapeutic agent also are known to those of skill in the art. Forexample, antibody-Pseudomonas exotoxin A fusion proteins have beendescribed by Chaudhary et al., Nature 339:394 (1989), Brinkmann et al.,Proc. Nat'l Acad. Sci. USA 88:8616 (1991), Batra et al., Proc. Nat'lAcad. Sci. USA 89:5867 (1992), Friedman et al., J. Immunol. 150:3054(1993), Wels et al., Int. J. Can. 60:137 (1995), Fominaya et al., J.Biol. Chem. 271:10560 (1996), Kuan et al., Biochemistry 35:2872 (1996),and Schmidt et al., Int. J. Can. 65:538 (1996). Antibody-toxin fusionproteins containing a diphtheria toxin moiety have been described byKreitman et al., Leukemia 7:553 (1993), Nicholls et al., J. Biol. Chem.268:5302 (1993), Thompson et al., J. Biol. Chem. 270:28037 (1995), andVallera et al., Blood 88:2342 (1996). Deonarain et al., Tumor Targeting1:177 (1995), have described an antibody-toxin fusion protein having anRNase moiety, while Linardou et al., Cell Biophys. 24-25:243 (1994),produced an antibody-toxin fusion protein comprising a DNase Icomponent. Gelonin was used as the toxin moiety in the antibody-toxinfusion protein of Wang et al., Abstracts of the 209th ACS NationalMeeting, Anaheim, Calif., Apr. 2-6, 1995, Part 1, BIOT005. As a furtherexample, Dohlsten et al., Proc. Nat'l Acad. Sci. USA 91:8945 (1994),reported an antibody-toxin fusion protein comprising Staphylococcalenterotoxin-A.

Illustrative of toxins which are suitably employed in the preparation ofsuch fusion proteins are ricin, abrin, ribonuclease, DNase I,Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin,diphtherin toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin. See,for example, Pastan et al., Cell 47:641 (1986), and Goldenberg, CA—ACancer Journal for Clinicians 44:43 (1994). Other suitable toxins areknown to those of skill in the art.

Antibodies of the present invention may also be used in ADEPT byconjugating the antibody to a prodrug-activating enzyme which converts aprodrug (e.g., a peptidyl chemotherapeutic agent, See WO81/01145) to anactive anti-cancer drug. See, for example, WO88/07378 and U.S. Pat. No.4,975,278.

The enzyme component of the immunoconjugate useful for ADEPT includesany enzyme capable of acting on a prodrug in such a way so as to covertit into its more active, cytotoxic form.

Enzymes that are useful in the this invention include, but are notlimited to: alkaline phosphatase; arylsulfatase; cytosine deaminase,5-fluorouracil; proteases, such as serratia protease, thermolysin,subtilisin, carboxypeptidases and cathepsins (such as cathepsins B andL); D-alanylcarboxypeptidases; carbohydrate-cleaving enzymes such asβ-galactosidase and neuraminidase; β-lactamase; and penicillin amidases,such as penicillin V amidase or penicillin G amidase. Alternatively,antibodies with enzymatic activity, also known in the art as abzymes,can be used to convert the prodrugs of the invention into free activedrugs (See, e.g., Massey, Nature 328: 457-458 (1987). Antibody-abzymeconjugates can be prepared as described herein for delivery of theabzyme to a tumor cell population.

The enzymes above can be covalently bound to the antibodies bytechniques well known in the art such as the use of theheterobifunctional crosslinking reagents discussed above. Alternatively,fusion proteins comprising at least the antigen binding region of anantibody of the invention linked to at least a functionally activeportion of an enzyme of the invention can be constructed usingrecombinant DNA techniques well known in the art (See, e.g., Neubergeret al., Nature 312: 604-608 (1984))

Preparing Amino Acid Sequence Variants

It is contemplated that modified polypeptide compositions comprisingone, two, three, four, five, and/or six CDRs of an antibody orpolypeptide binding agent are generated, wherein a CDR or non-CDR regionis altered to provide increased specificity or affinity to the antigen,or to provide increased modulation of binding affinity between thetarget and its signaling partner. For example, sites within antibodyCDRs are typically modified in series, e.g., by substituting first withconservative choices (e.g., hydrophobic amino acid substituted for anon-identical hydrophobic amino acid) and then with more dissimilarchoices (e.g., hydrophobic amino acid substituted for a charged aminoacid), and then deletions or insertions may be made at the targetedsite. It is contemplated that conservative substitutions within the CDRallow the variable region to retain biological activity. For example,using the conserved framework sequences surrounding the CDRs, PCRprimers complementary to these consensus sequences are generated toamplify the antigen-specific CDR sequence located between the primerregions. Techniques for cloning and expressing nucleotide andpolypeptide sequences are well-established in the art [see e.g. Sambrooket al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold SpringHarbor, N.Y. (1989)]. The amplified CDR sequences are ligated into anappropriate plasmid. The plasmid comprising one, two, three, four, fiveand/or six cloned CDRs optionally contains additional polypeptideencoding regions linked to the CDR.

Polypeptide binding agents comprising the modified CDRs are screened forbinding affinity for the original antigen. Additionally, the antibody orpolypeptide is further tested for its ability to neutralize the activityof its antigen. For example, antibodies of the invention may be analyzedas set out in the Examples to determine their ability to interfere withthe biological activity of the target.

Modifications may be made by conservative or non-conservative amino acidsubstitutions described in greater detail below. “Insertions” or“deletions” are preferably in the range of about 1 to 20 amino acids,more preferably 1 to 10 amino acids. The variation may be introduced bysystematically making substitutions of amino acids in an antibodypolypeptide molecule using recombinant DNA techniques and assaying theresulting recombinant variants for activity. Nucleic acid alterationscan be made at sites that differ in the nucleic acids from differentspecies (variable positions) or in highly conserved regions (constantregions). Methods for altering antibody sequences and expressingantibody polypeptide compositions useful in the invention are describedin greater detail below.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intra-sequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue or the antibody(including antibody fragment) fused to an epitope tag or a salvagereceptor epitope. Other insertional variants of the antibody moleculeinclude the fusion to a polypeptide which increases the serum half-lifeof the antibody, e.g. at the N-terminus or C-terminus.

The term “epitope tagged” refers to the antibody fused to an epitopetag. The epitope tag polypeptide has enough residues to provide anepitope against which an antibody there against can be made, yet isshort enough such that it does not interfere with activity of theantibody. The epitope tag preferably is sufficiently unique so that theantibody there against does not substantially cross-react with otherepitopes. Suitable tag polypeptides generally have at least 6 amino acidresidues and usually between about 8-50 amino acid residues (preferablybetween about 9-30 residues). Examples include the flu hemagglutinin(HA) tag polypeptide and its antibody 12CA5 (Field et al., Mol. Cell.Biol. 8: 2159-2165 (1988)); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7and 9E10 antibodies thereto (Evan et al., Mol. Cell. Biol. 5:3610-16(1985)); and the Herpes Simplex virus glycoprotein D (gD) tag and itsantibody (Paborsky et al., Protein Engineering 3:547-53 (1990)). Otherexemplary tags are a poly-histidine sequence, generally around sixhistidine residues, that permits isolation of a compound so labeledusing nickel chelation. Other labels and tags, such as the FLAG® tag(Eastman Kodak, Rochester, N.Y.), well known and routinely used in theart, are embraced by the invention.

As used herein, the term “salvage receptor binding epitope” refers to anepitope of the Fc region of an IgG molecule (e.g., IgG₁, IgG₂, IgG₃, orIgG₄) that is responsible for increasing the in vivo serum half-life ofthe IgG molecule.

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the antibody moleculeremoved and a different residue inserted in its place. Substitutionalmutagenesis within any of the hypervariable or CDR regions or frameworkregions is contemplated. Conservative substitutions involve replacing anamino acid with another member of its class. Non-conservativesubstitutions involve replacing a member of one of these classes with amember of another class.

Conservative amino acid substitutions are made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues involved.For example, nonpolar (hydrophobic) amino acids include alanine (Ala,A), leucine (Leu, L), isoleucine (Ile, I), valine (Val, V), proline(Pro, P), phenylalanine (Phe, F), tryptophan (Trp, W), and methionine(Met, M); polar neutral amino acids include glycine (Gly, G), serine(Ser, S), threonine (Thr, T), cysteine (Cys, C), tyrosine (Tyr, Y),asparagine (Asn, N), and glutamine (Gln, Q); positively charged (basic)amino acids include arginine (Arg, R), lysine (Lys, K), and histidine(His, H); and negatively charged (acidic) amino acids include asparticacid (Asp, D) and glutamic acid (Glu, E).

Any cysteine residue not involved in maintaining the proper conformationof the antibody also may be substituted, generally with serine, toimprove the oxidative stability of the molecule and prevent aberrantcrosslinking Conversely, cysteine bond(s) may be added to the antibodyto improve its stability (particularly where the antibody is an antibodyfragment such as an Fv fragment).

Affinity Maturation

Affinity maturation generally involves preparing and screening antibodyvariants that have substitutions within the CDRs of a parent antibodyand selecting variants that have improved biological properties such asstronger binding affinity relative to the parent antibody. A convenientway for generating such substitutional variants is affinity maturationusing phage display. Briefly, several hypervariable region sites (e.g.6-7 sites) are mutated to generate all possible amino substitutions ateach site. The antibody variants thus generated are displayed in amonovalent fashion from filamentous phage particles as fusions to thegene III product of M13 packaged within each particle. Thephage-displayed variants are then screened for their biological activity(e.g. binding affinity). See e.g., WO 92/01047, WO 93/112366, WO95/15388 and WO 93/19172.

Current antibody affinity maturation methods belong to two mutagenesiscategories: stochastic and nonstochastic. Error prone PCR, mutatorbacterial strains (Low et al., J. Mol. Biol. 260, 359-68 (1996)), andsaturation mutagenesis (Nishimiya et al., J. Biol. Chem. 275:12813-20(2000); Chowdhury, P. S. Methods Mol. Biol. 178, 269-85 (2002)) aretypical examples of stochastic mutagenesis methods (Rajpal et al., ProcNatl Acad Sci USA. 102:8466-71 (2005)). Nonstochastic techniques oftenuse alanine-scanning or site-directed mutagenesis to generate limitedcollections of specific variants. Some methods are described in furtherdetail below.

Affinity Maturation Via Panning Methods—

Affinity Maturation of Recombinant antibodies is commonly performedthrough several rounds of panning of candidate antibodies in thepresence of decreasing amounts of antigen. Decreasing the amount ofantigen per round selects the antibodies with the highest affinity tothe antigen thereby yielding antibodies of high affinity from a largepool of starting material. Affinity maturation via panning is well knownin the art and is described, for example, in Huls et al. (Cancer ImmunolImmunother. 50:163-71 (2001)). Methods of affinity maturation usingphage display technologies are described elsewhere herein and known inthe art (see e.g., Daugherty et al., Proc Natl Acad Sci USA. 97:2029-34(2000)).

Look-Through Mutagenesis—

Look-through mutagenesis (LTM) (Rajpal et al., Proc Natl Acad Sci USA.102:8466-71 (2005)) provides a method for rapidly mapping theantibody-binding site. For LTM, nine amino acids, representative of themajor side-chain chemistries provided by the 20 natural amino acids, areselected to dissect the functional side-chain contributions to bindingat every position in all six CDRs of an antibody. LTM generates apositional series of single mutations within a CDR where each “wildtype” residue is systematically substituted by one of nine selectedamino acids. Mutated CDRs are combined to generate combinatorialsingle-chain variable fragment (scFv) libraries of increasing complexityand size without becoming prohibitive to the quantitative display of allvariants. After positive selection, clones with stronger bindingaffinity are sequenced, and beneficial mutations are mapped.

Error Prone PCR—

Error-prone PCR involves the randomization of nucleic acids betweendifferent selection rounds. The randomization occurs at a low rate bythe intrinsic error rate of the polymerase used but can be enhanced byerror-prone PCR (Zaccolo et al., J. Mol. Biol. 285:775-783 (1999)) usinga polymerase having a high intrinsic error rate during transcription(Hawkins et al., J Mol Biol. 226:889-96 (1992)). After the mutationcycles, clones with stronger binding affinity for the antigen areselected using routine methods in the art.

DNA Shuffling—

Nucleic acid shuffling is a method for in vitro or in vivo homologousrecombination of pools of shorter or smaller polynucleotides to producevariant polynucleotides. DNA shuffling has been described in U.S. Pat.No. 6,605,449, U.S. Pat. No. 6,489,145, WO 02/092780 and Stemmer, Proc.Natl. Acad. Sci. USA, 91:10747-51 (1994). Generally, DNA shuffling iscomprised of 3 steps: fragmentation of the genes to be shuffled withDNase I, random hybridization of fragments and reassembly or filling inof the fragmented gene by PCR in the presence of DNA polymerase (sexualPCR), and amplification of reassembled product by conventional PCR.

DNA shuffling differs from error-prone PCR in that it is an inversechain reaction. In error-prone PCR, the number of polymerase start sitesand the number of molecules grows exponentially. In contrast, in nucleicacid reassembly or shuffling of random polynucleotides the number ofstart sites and the number (but not size) of the random polynucleotidesdecreases over time.

In the case of an antibody, DNA shuffling allows the free combinatorialassociation of all of the CDR1s with all of the CDR2s with all of theCDR3s, for example. It is contemplated that multiple families ofsequences can be shuffled in the same reaction. Further, shufflinggenerally conserves the relative order, such that, for example, CDR1will not be found in the position of CDR2. Rare shufflants will containa large number of the best (e.g. highest affinity) CDRs and these rareshufflants may be selected based on their superior affinity.

The template polynucleotide which may be used in DNA shuffling may beDNA or RNA. It may be of various lengths depending on the size of thegene or shorter or smaller polynucleotide to be recombined orreassembled. Preferably, the template polynucleotide is from 50 by to 50kb. The template polynucleotide often should be double-stranded.

It is contemplated that single-stranded or double-stranded nucleic acidpolynucleotides having regions of identity to the templatepolynucleotide and regions of heterology to the template polynucleotidemay be added to the template polynucleotide, during the initial step ofgene selection. It is also contemplated that two different but relatedpolynucleotide templates can be mixed during the initial step.

Alanine Scanning—

Alanine scanning mutagenesis can be performed to identify hypervariableregion residues that contribute significantly to antigen binding.Cunningham and Wells, (Science 244:1081-1085 (1989)). A residue or groupof targeted residues are identified (e.g., charged residues such as arg,asp, his, lys, and glu) and replaced by a neutral or negatively chargedamino acid (most preferably alanine or polyalanine) to affect theinteraction of the amino acids with antigen. Those amino acid locationsdemonstrating functional sensitivity to the substitutions then arerefined by introducing further or other variants at, or for, the sitesof substitution.

Computer-Aided Design—

Alternatively, or in addition, it may be beneficial to analyze a crystalstructure of the antigen-antibody complex to identify contact pointsbetween the antibody and antigen, or to use computer software to modelsuch contact points. Such contact residues and neighboring residues arecandidates for substitution according to the techniques elaboratedherein. Once such variants are generated, the panel of variants issubjected to screening as described herein and antibodies with superiorproperties in one or more relevant assays may be selected for furtherdevelopment.

Formulation of Pharmaceutical Compositions

To administer polypeptide binding agents of the invention to human ortest mammals, it is preferable to formulate the polypeptide bindingagent in a sterile composition comprising one or more sterilepharmaceutically acceptable carriers. The phrase “pharmaceutically orpharmacologically acceptable” refer to molecular entities andcompositions that do not produce allergic, or other adverse reactionswhen administered using routes well-known in the art, as describedbelow. “Pharmaceutically acceptable carriers” include any and allclinically useful solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents and thelike.

The polypeptide binding agent is administered by any suitable means,including parenteral, subcutaneous, intraperitoneal, intrapulmonary, andintranasal, and, if desired for local treatment, intralesionaladministration. Parenteral infusions include intravenous, intraarterial,intraperitoneal, intramuscular, intradermal or subcutaneousadministration. Preferably the dosing is given by injections, mostpreferably intravenous or subcutaneous injections, depending in part onwhether the administration is brief or chronic. Other administrationmethods are contemplated, including topical, particularly transdermal,transmucosal, rectal, oral or local administration e.g. through acatheter placed close to the desired site.

Pharmaceutical compositions of the present invention containing apolypeptide binding agent of the invention as an active ingredient maycontain sterile pharmaceutically acceptable carriers or additivesdepending on the route of administration. Examples of such carriers oradditives include water, a pharmaceutical acceptable organic solvent,collagen, polyvinyl alcohol, polyvinylpyrrolidone, a carboxyvinylpolymer, carboxymethylcellulose sodium, polyacrylic sodium, sodiumalginate, water-soluble dextran, carboxymethyl starch sodium, pectin,methyl cellulose, ethyl cellulose, xanthan gum, gum Arabic, casein,gelatin, agar, diglycerin, glycerin, propylene glycol, polyethyleneglycol, Vaseline, paraffin, stearyl alcohol, stearic acid, human serumalbumin (HSA), mannitol, sorbitol, lactose, a pharmaceuticallyacceptable surfactant and the like. Additives used are chosen from, butnot limited to, the above or combinations thereof, as appropriate,depending on the dosage form of the present invention. For solutions oremulsions, suitable carriers include, for example, aqueous oralcoholic/aqueous solutions, emulsions or suspensions, including salineand buffered media. Parenteral vehicles can include sodium chloridesolution, Ringer's dextrose, dextrose and sodium chloride, lactatedRinger's or fixed oils. Intravenous vehicles can include variousadditives, preservatives, or fluid, nutrient or electrolytereplenishers. A variety of aqueous carriers are suitable, e.g., sterilephosphate buffered saline solutions, bacteriostatic water, water,buffered water, 0.4% saline, 0.3% glycine, and the like, and may includeother proteins for enhanced stability, such as albumin, lipoprotein,globulin, etc., subjected to mild chemical modifications or the like.

Therapeutic formulations of the polypeptide binding agent are preparedfor storage by mixing the polypeptide binding agent having the desireddegree of purity with optional physiologically acceptable carriers,excipients or stabilizers (Remington's Pharmaceutical Sciences 16thedition, Osol, A. Ed. (1980)), in the form of lyophilized formulationsor aqueous solutions. Acceptable carriers, excipients, or stabilizersare nontoxic to recipients at the dosages and concentrations employed,and include buffers such as phosphate, citrate, succinate and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

The active ingredients may also be entrapped in microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Aqueous suspensions may contain the active compound in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents may be a naturally-occurring phosphatide,for example lecithin, or condensation products of an alkylene oxide withfatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyl-eneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions may also contain one or more preservatives, forexample ethyl, or n-propyl, p-hydroxybenzoate.

The antibodies of the invention can be lyophilized for storage andreconstituted in a suitable carrier prior to use. This technique hasbeen shown to be effective with conventional immunoglobulins. Anysuitable lyophilization and reconstitution techniques can be employed.It will be appreciated by those skilled in the art that lyophilizationand reconstitution can lead to varying degrees of antibody activity lossand that use levels may have to be adjusted to compensate.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active compound inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.

The concentration of polypeptide binding agent in these formulations canvary widely, for example from less than about 0.5%, usually at or atleast about 1% to as much as 15 or 20% by weight and will be selectedprimarily based on fluid volumes, viscosities, etc., in accordance withthe particular mode of administration selected. Thus, a typicalpharmaceutical composition for parenteral injection could be made up tocontain 1 ml sterile buffered water, and 50 mg of polypeptide bindingagent. A typical composition for intravenous infusion could be made upto contain 250 ml of sterile Ringer's solution, and 150 mg ofpolypeptide binding agent. Actual methods for preparing parenterallyadministrable compositions will be known or apparent to those skilled inthe art and are described in more detail in, for example, Remington'sPharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pa.(1980). An effective dosage of polypeptide binding agent is within therange of 0.01 mg to 1000 mg per kg of body weight per administration.

The pharmaceutical compositions may be in the form of a sterileinjectable aqueous, oleaginous suspension, dispersions or sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersions. The suspension may be formulated according tothe known art using those suitable dispersing or wetting agents andsuspending agents which have been mentioned above. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example as a solution in 1,3-butane diol. The carrier can be asolvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), suitable mixtures thereof, vegetable oils,Ringer's solution and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose any bland fixed oil may be employedincluding synthetic mono- or diglycerides. In addition, fatty acids suchas oleic acid find use in the preparation of injectables.

In all cases the form must be sterile and must be fluid to the extentthat easy syringability exists. The proper fluidity can be maintained,for example, by the use of a coating, such as lecithin, by themaintenance of the required particle size in the case of dispersion andby the use of surfactants. It must be stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms, such as bacteria and fungi. The prevention ofthe action of microorganisms can be brought about by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In manycases, it will be desirable to include isotonic agents, for example,sugars or sodium chloride. Prolonged absorption of the injectablecompositions can be brought about by the use in the compositions ofagents delaying absorption, for example, aluminum monostearate andgelatin.

Compositions useful for administration may be formulated with uptake orabsorption enhancers to increase their efficacy. Such enhancers includefor example, salicylate, glycocholate/linoleate, glycholate, aprotinin,bacitracin, SDS, caprate and the like. See, e.g., Fix (J. Pharm. Sci.,85:1282-1285 (1996)) and Oliyai and Stella (Ann. Rev. Pharmacol.Toxicol., 32:521-544 (1993)).

Biophysical Assays

Complex biological events can be studied via molecular biophysicalapproaches which consider them as systems of interacting units which canbe understood in terms of statistical mechanics, thermodynamics andchemical kinetics

In certain embodiments, the assays of the present invention may employ adetectable moiety. The detectable moiety can be any one which is capableof producing, either directly or indirectly, a measurable signal, suchas a radioactive, chromogenic, luminescence, or fluorescent signal,which can be used to quantitate the amount of bound detectable moiety orlabel in a sample. Detectable labels known in the art includeradioisotopes, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I,electrochemiluminescent labels (such as Ruthenium (Ru)-based catalyst inconjunction with substrates, etc.), luminescent or bioluminescent labels(e.g., Europium, Vanadium), fluorescent or chemiluminescent compounds,such as fluorescein isothiocyanate, rhodamine, or luciferin, enzymes(e.g., enzyme, such as alkaline phosphatase, β-galactosidase, orhorseradish peroxidase), colorimetric labels such as colloidal gold,colored glass or plastic beads (e.g., polystyrene, polypropylene, latex,etc.), paramagnetic atoms or magnetic agents, electron-dense reagents, anano- or micro-bead containing a fluorescent dye, nanocrystals, aquantum dot, a quantum bead, a nanotag, dendrimers with a fluorescentlabel, a micro-transponder, an electron donor molecule or molecularstructure, or a light reflecting particle. the microparticles may benanocrystals or quantum dots. Nanocrystals are substances that absorbphotons of light, then re-emit photons at a different wavelength(fluorophores). In addition, additional florescent labels, or secondaryantibodies may be conjugated to the nanocrystals. Nanocrystals arecommercially available from sources such as Invitrogen and EvidentTechnologies (Troy, N.Y.). Other labels includeE)-5-[2-(methoxycarbonyl)ethenyl]cytidine, which is a nonfluorescentmolecule that when subjected to ultraviolet (UV) irradiation yields aproduct, 3-.beta.-D-ribofuranosyl-2,7-dioxopyrido[2,3-d]pyrimidine,which displays a strong fluorescence signal. Bar code labels aredescribed in U.S. Patent Publication No. US 20070037195.

A variety of assay methods known in the art may be employed in thepresent invention, such as competitive binding assays, direct andindirect sandwich assays, immunoprecipitation assays, fluorescentresonance energy transfer (FRET), electroimmunoassays surface plasmonresonance (SPR), and nanoparticle-derived techniques

Competitive binding assays rely on the ability of a labeled standard(e.g., an antigen or a fragment thereof to which a polypeptide bindingagent binds) to compete with antigen in the test sample for binding tothe polypeptide binding agent. The amount of antigen in the test sampleis inversely proportional to the amount of standard that becomes boundto the antibodies. To facilitate determining the amount of standard thatbecomes bound, the antibodies typically are insolubilized before orafter the competition, so that the bound antigen may conveniently beseparated from the unbound antigen. In alternative embodiments,competitive binding assays measure the ability of a labeled polypeptidebinding agent to compete with unlabeled polypeptide binding agent forbinding to antigen or a fragment thereof.

Sandwich assays typically involve the use of two antibodies, eachcapable of binding to a different immunogenic portion, or epitope, ofthe protein to be detected and/or quantitated. In a sandwich assay, theanalyte in the test sample is typically bound by a first polypeptidebinding agent which is immobilized on a solid phase, and thereafter asecond polypeptide binding agent binds to the analyte, thus forming aninsoluble three-part complex. See, e.g., U.S. Pat. No. 4,376,110. Thesecond polypeptide binding agent may itself be labeled with a detectablemoiety (direct sandwich assays) or may be measured using ananti-immunoglobulin antibody that is labeled with a detectable moiety(indirect sandwich assays). For example, one type of sandwich assay isan enzyme-linked immunosorbent assay (ELISA), in which case thedetectable moiety is an enzyme. See, for example, chapter 18, CurrentProtocols in Molecular Biology, Ausubel et al., eds., John Wiley & Sons,New York, N.Y. (1995).

Yet another example of an assay method involves fluorescent resonanceenergy transfer (FRET) emissions. For example, one compound is labeledwith a FRET donor molecule and its binding partner is labeled with aFRET acceptor molecule, or vice versa. When binding occurs between thebinding partners, the FRET donor and FRET acceptor molecules are broughtinto proximity and emit fluorescence at a certain wavelength. A narrowband pass filter can be used to block all wavelengths except that of thelabel. FRET molecule pairs are commercially available in the art (e.g.,from Invitrogen), and may be used according to the manufacturer'sprotocol. FRET emissions are detected using optical imaging techniques,such as a CCD camera.

Yet another example of an assay method is bioluminescence resonanceenergy transfer (BRET), for example using biosensors as described inWO/06/086883.

Another type of assay involves labeling with an electron donor. Onemolecule is labeled with an electron donor and the interacting moleculeis bound to an electrical contact, or vice versa. When binding occursbetween the binding partners, the label donates electrons to theelectrical contact. See, for example, Ghindilis, Biochem Soc Trans.28:84-9, (2000) and Dai et al., Cancer Detect Prev. 29:233-40 (2005),which describe methods for electro immunoassays. The electron contactwould then be read by an A to D (analog to digital) converter andquantified. The higher the electron count the more interactions tookplace.

One embodiment of a label capable of single molecule detection is theuse of plasmon-resonant particles (PRPs) as optical reporters, asdescribed in Schultz et al., Proc. Natl. Acad. Sci. USA 97:996-1001(2000), incorporated herein by reference. PRPs are metallicnanoparticles, e.g. 40-100 nm in diameter, which scatter light becauseof a collective resonance of the conduction electrons in the metal(i.e., the surface plasmon resonance). The magnitude, peak wavelength,and spectral bandwidth of the plasmon resonance associated with ananoparticle are dependent on the particle's size, shape, and materialcomposition, as well as the local environment. By influencing theseparameters during preparation, PRPs can be formed that have scatteringpeak anywhere in the visible range of the spectrum. For spherical PRPs,both the peak scattering wavelength and scattering efficiency increasewith larger radius, providing a means for producing differently coloredlabels. Populations of silver spheres, for example, can be reproduciblyprepared for which the peak scattering wavelength is within a fewnanometers of the targeted wavelength, by adjusting the final radius ofthe spheres during preparation. Because PRPs are bright, yet nano sized,they are used as indicators for single-molecule detection; that is, thepresence of a bound PRP in a field of view can indicate a single bindingevent. An example of a surface plasmon resonance detector system is theBIAcore assay system. See, e.g., Malmquist, J Molec Recognition, 7:1-7(1994).

Molecular interactions may also be detected using nanoparticle-derivedtechniques. See, for example, Ao et al., Anal Chem. 78:1104-6 (2006),which describes gold nanoparticle quenching, Tang et al., BiosensBioelectron. 2005 Nov. 30, which describes SiO(2)/Au nanoparticlesurfaces in antibody detection, and Lieu et al., J Immunol Methods.307:34-40 (2005), which describes silicon dioxide nanoparticlescontaining dibromofluorescein for use in solid substrate-roomtemperature phosphorescence immunoassay (SS-RTP-IA).

A KinExA assay is also useful to measure the affinity of a modulatingantibody for its antigen. An exemplary KinExA assay is described inExample 20. For example, a KinExA assay measures very low levels ofligand in cell culture media. This assay allows the binding of ligand tocells expressing the cognate receptor to be measured by determining thelevel of ligand depletion from the cell culture media. As the ligandbecomes bound to the cells, the concentration of ligand in the cellculture media drops. By using a titration of cells expressing thereceptor and measuring the percent free ligand, the affinity of theligand-receptor interaction is estimated using KinExA software(Sāpidyne, Boise Id.). This assay is used to measure the degree ofmodulation of ligand binding activity shown by various anti-receptorantibodies.

Any of the preceding measurements of binding affinity or binding rateparameters may be carried out in assays where one or more of the firstcomponent, second component and polypeptide binding agent are insolution, or in assays where one or more of the first component, secondcomponent and polypeptide binding agent are linked to a solid phase(covalently or noncovalently), or in assays where one or more of thefirst component, second component and polypeptide binding agent areexpressed on a cell surface. The first and/or second components may eachthemselves be complexes of multiple compounds.

Administration and Dosing

In one aspect, methods of the invention include a step of administeringa pharmaceutical composition.

Methods of the invention are performed using any medically-acceptedmeans for introducing a therapeutic directly or indirectly into amammalian subject, including but not limited to injections, oralingestion, intranasal, topical, transdermal, parenteral, inhalationspray, vaginal, or rectal administration. The term parenteral as usedherein includes subcutaneous, intravenous, intramuscular, andintracisternal injections, as well as catheter or infusion techniques.Administration by, intradermal, intramammary, intraperitoneal,intrathecal, retrobulbar, intrapulmonary injection and or surgicalimplantation at a particular site is contemplated as well. Suitabledelivery devices may include those developed for the delivery of insulin(see e.g. Owens et al Diabetic Med. 20(11):886-898, 2003).

In one embodiment, administration is performed at the site of a canceror affected tissue needing treatment by direct injection into the siteor via a sustained delivery or sustained release mechanism, which candeliver the formulation internally. For example, biodegradablemicrospheres or capsules or other biodegradable polymer configurationscapable of sustained delivery of a composition (e.g., a solublepolypeptide, antibody, or small molecule) can be included in theformulations of the invention implanted at the site.

Therapeutic compositions may also be delivered to the patient atmultiple sites. The multiple administrations may be renderedsimultaneously or may be administered over a period of time. In certaincases it is beneficial to provide a continuous flow of the therapeuticcomposition. Additional therapy may be administered on a period basis,for example, hourly, daily, weekly, every 2 weeks, every 3 weeks, ormonthly.

Also contemplated in the present invention is the administration ofmultiple agents, such as an antibody composition in conjunction with asecond agent as described herein.

The amounts of antibody composition in a given dosage will varyaccording to the size of the individual to whom the therapy is beingadministered as well as the characteristics of the disorder beingtreated. In exemplary treatments, it may be necessary to administerabout 1 mg/day, 5 mg/day, 10 mg/day, 20 mg/day, 50 mg/day, 75 mg/day,100 mg/day, 150 mg/day, 200 mg/day, 250 mg/day, 500 mg/day or 1000mg/day. These concentrations may be administered as a single dosage formor as multiple doses. Standard dose-response studies, first in animalmodels and then in clinical testing, reveal optimal dosages forparticular disease states and patient populations.

Combination Therapy

It one embodiment, an antibody of the invention is administered with asecond agent useful to treat a disease or disorder as described herein.It is contemplated that two or more antibodies to different epitopes ofthe target antigen may be mixed such that the combination of antibodiestogether to provide still improved efficacy against a condition ordisorder to be treated associated with the target polypeptide.Compositions comprising one or more antibody of the invention may beadministered to persons or mammals suffering from, or predisposed tosuffer from, a condition or disorder to be treated associated with thetarget polypeptide.

Concurrent administration of two therapeutic agents does not requirethat the agents be administered at the same time or by the same route,as long as there is an overlap in the time period during which theagents are exerting their therapeutic effect. Simultaneous or sequentialadministration is contemplated, as is administration on different daysor weeks.

A second agent may be other therapeutic agents, such as anti-diabeticagents, cytokines, growth factors, other anti-inflammatory agents,anti-coagulant agents, agents that will lower or reduce blood pressure,agents that will reduce cholesterol, triglycerides, LDL, VLDL, orlipoprotein(a) or increase HDL, agents that will increase or decreaselevels of cholesterol-regulating proteins, anti-neoplastic drugs ormolecules. For patients with a hyperproliferative disorder, such ascancer or a tumor, combination with second therapeutic modalities suchas radiotherapy, chemotherapy, photodynamic therapy, or surgery is alsocontemplated.

Exemplary anti-diabetic agents include, but are not limited to, 1)sulfonylureas (e.g., glimepiride, glisentide, sulfonylurea, AY31637); 2)biguanides (e.g., metformin); 3) alpha-glucosidase inhibitors (e.g.,acarbose, miglitol); 4) thiazol-idinediones (e.g., troglitazone,pioglitazone, rosiglitazone, glipizide, balaglitazone, rivoglitazone,netoglitazone, troglitazone, englitazone, AD 5075, T 174, YM 268, R102380, NC 2100, NIP 223, NIP 221, MK 0767, ciglitazone, adaglitazone,CLX 0921, darglitazone, CP 92768, BM 152054); 5) glucagon-like-peptides(GLP) and GLP analogs or agonists of GLP-1 receptor (e.g. exendin) orstabilizers thereof (e.g. DPP4 inhibitors, such as sitagliptin); and 6)insulin or analogues or mimetics thereof (e.g. LANTUS®).

It is contemplated the antibody of the invention and the second agentmay be given simultaneously, in the same formulation. It is furthercontemplated that the agents are administered in a separate formulationand administered concurrently, with concurrently referring to agentsgiven within 30 minutes of each other.

In another aspect, the second agent is administered prior toadministration of the antibody composition. Prior administration refersto administration of the second agent within the range of one week priorto treatment with the antibody, up to 30 minutes before administrationof the antibody. It is further contemplated that the second agent isadministered subsequent to administration of the antibody composition.Subsequent administration is meant to describe administration from 30minutes after antibody treatment up to one week after antibodyadministration.

It is further contemplated that other adjunct therapies may beadministered, where appropriate. For example, the patient may also beadministered a diabetic diet or food plan, surgical therapy, orradiation therapy where appropriate.

It will also be apparent that dosing may be modified if traditionaltherapeutics are administered in combination with therapeutics of theinvention.

Methods of Use

Therapeutic Indications for INSR Agonists/Positive Modulators

In another embodiment, the invention provides a method for inhibitingtarget activity by administering a target-specific antibody to a patientin need thereof. Any of the types of antibodies described herein may beused therapeutically. In exemplary embodiments, the target specificantibody is a human, chimeric or humanized antibody. In anotherexemplary embodiment, the target is human and the patient is a humanpatient. Alternatively, the patient may be a mammal that expresses atarget protein that the target specific antibody cross-reacts with. Theantibody may be administered to a non-human mammal expressing a targetprotein with which the antibody cross-reacts (i.e. a primate) forveterinary purposes or as an animal model of human disease. Such animalmodels may be useful for evaluating the therapeutic efficacy of targetspecific antibodies of the invention.

Insulin resistance describes a condition in which physiological amountsof insulin are inadequate to produce a normal insulin response fromcells or tissues. Insulin resistance is associated with a number ofdisease states and conditions and is present in approximately 30-40% ofnon-diabetic individuals. These disease states and conditions include,but are not limited to, pre-diabetes, metabolic syndrome (also referredto as insulin resistance syndrome), Type 2 diabetes mellitus, polycysticovary disease (PCOS) and non-alcoholic fatty liver disease (NAFLD)(reviewed in Woods et al, End, Metab & Immune Disorders—Drug Targets 9:187-198, 2009).

Pre-diabetes is a state of abnormal glucose tolerance characterized byeither impaired glucose tolerance (IGT) or impaired fasting glucose(IFG). Patients with pre-diabetes are insulin resistant and are at highrisk for future progression to overt Type 2 diabetes. Metabolic syndromeis an associated cluster of traits that include, but is not limited to,hyperinsulinemia, abnormal glucose tolerance, obesity, redistribution offat to the abdominal or upper body compartment, hypertension,dysfibrinolysis, and a dyslipidemia characterized by high triglycerides,low HDL-cholesterol, and small dense LDL particles. Insulin resistancehas been linked to each of the traits, suggesting metabolic syndrome andinsulin resistance are intimately related to one another. The diagnosisof metabolic syndrome is a powerful risk factor for future developmentof Type 2 diabetes, as well as accelerated atherosclerosis resulting inheart attacks, strokes, and peripheral vascular disease.

Diabetes mellitus is a metabolic disorder in humans with a prevalence ofapproximately one percent in the general population (Foster, D. W.,Harrison's Principles of Internal Medicine, Chap. 114, pp. 661-678, 10thEd., McGraw-Hill, New York). The disease manifests itself as a series ofhormone-induced metabolic abnormalities that eventually lead to serious,long-term and debilitating complications involving several organ systemsincluding the eyes, kidneys, nerves, and blood vessels. Pathologically,the disease is characterized by lesions of the basement membranes,demonstrable under electron microscopy. Diabetes mellitus can be dividedinto two clinical syndromes, Type 1 and Type 2 diabetes mellitus.

Type 1, or insulin-dependent diabetes mellitus (IDDM), also referred toas the juvenile onset form, is a chronic autoimmune diseasecharacterized by the extensive loss of beta cells in the pancreaticIslets of Langerhans, which produce insulin. As these cells areprogressively destroyed, the amount of secreted insulin decreases,eventually leading to hyperglycemia (abnormally high level of glucose inthe blood) when the amount of secreted insulin drops below the normallyrequired blood glucose levels. Although the exact trigger for thisimmune response is not known, patients with IDDM have high levels ofantibodies against proteins expressed in pancreatic beta cells. However,not all patients with high levels of these antibodies develop IDDM. Type1 diabetics characteristically show very low or immeasurable plasmainsulin with elevated glucagon. Regardless of what the exact etiologyis, most Type 1 patients have circulating antibodies directed againsttheir own pancreatic cells including antibodies to insulin, to Islet ofLangerhans cell cytoplasm and to the enzyme glutamic acid decarboxylase.An immune response specifically directed against beta cells (insulinproducing cells) leads to Type 1 diabetes. The current treatment forType 1 diabetic patients is the injection of insulin, and may alsoinclude modifications to the diet in order to minimize hyperglycemiaresulting from the lack of natural insulin, which in turn, is the resultof damaged beta cells. Diet is also modified with regard to insulinadministration to counter the hypoglycemic effects of the hormone.

Type 2 diabetes (also referred to as non-insulin dependent diabetesmellitus (NIDDM), maturity onset form, adult onset form) develops whenmuscle, fat and liver cells fail to respond normally to insulin. Thisfailure to respond (called insulin resistance) may be due to reducednumbers of insulin receptors on these cells, or a dysfunction ofsignaling pathways within the cells, or both. The beta cells initiallycompensate for this insulin resistance by increasing insulin output.Over time, these cells become unable to produce enough insulin tomaintain normal glucose levels, indicating progression to Type 2diabetes. Type 2 diabetes is brought on by a combination of genetic andacquired risk factors, including a high-fat diet, lack of exercise, andaging. Greater than 90% of the diabetic population suffers from Type 2diabetes and the incidence continues to rise, becoming a leading causeof mortality, morbidity and healthcare expenditure throughout the world(Amos et al., Diabetic Med. 14:S1-85, 1997).

Type 2 diabetes is a complex disease characterized by defects in glucoseand lipid metabolism. Typically there are perturbations in manymetabolic parameters including increases in fasting plasma glucoselevels, free fatty acid levels and triglyceride levels, as well as adecrease in the ratio of HDL/LDL. As discussed above, one of theprincipal underlying causes of diabetes is thought to be an increase ininsulin resistance in peripheral tissues, principally muscle and fat.The causes of Type 2 diabetes are not well understood. It is thoughtthat both resistance of target tissues to the action of insulin anddecreased insulin secretion (“β-cell failure”) occur. Majorinsulin-responsive tissues for glucose homeostasis are liver, in whichinsulin stimulates glycogen synthesis and inhibits gluconeogenesis;muscle, in which insulin stimulates glucose uptake and glycogenstimulates glucose uptake and inhibits lipolysis. Thus, as a consequenceof the diabetic condition, there are elevated levels of glucose in theblood, which can lead to glucose-mediated cellular toxicity andsubsequent morbidity (nephropathy, neuropathy, retinopathy, etc.).Insulin resistance is strongly correlated with the development of Type 2diabetes.

Currently, there are various pharmacological approaches for thetreatment of Type 2 diabetes (Scheen et al, Diabetes Care,22(9):1568-1577, 1999; Zangeneh et al, Mayo Clin. Proc. 78: 471-479,2003; Mohler et al, Med Res Rev 29(1): 125-195, 2009). They act viadifferent modes of action: 1) sulfonylureas (e.g., glimepiride,glisentide, sulfonylurea, AY31637) essentially stimulate insulinsecretion; 2) biguanides (e.g., metformin) act by promoting glucoseutilization, reducing hepatic glucose production and diminishingintestinal glucose output; 3) alpha-glucosidase inhibitors (e.g.,acarbose, miglitol) slow down carbohydrate digestion and consequentlyabsorption from the gut and reduce postprandial hyperglycemia; 4)thiazol-idinediones (e.g., troglitazone, pioglitazone, rosiglitazone,glipizide, balaglitazone, rivoglitazone, netoglitazone, troglitazone,englitazone, AD 5075, T 174, YM 268, R 102380, NC 2100, NIP 223, NIP221, MK 0767, ciglitazone, adaglitazone, CLX 0921, darglitazone, CP92768, BM 152054) enhance insulin action, thus promoting glucoseutilization in peripheral tissues; 5) glucagon-like-peptides andagonists (e.g. exendin) or stabilizers thereof (e.g. DPP4 inhibitors,such as sitagliptin) potentiate glucose-stimulated insulin secretion;and 6) insulin or analogues thereof (e.g. LANTUS®) stimulate tissueglucose utilization and inhibits hepatic glucose output. The abovementioned pharmacological approaches may be utilized individually or incombination therapy. However, each approach has its limitations andadverse effects. Over time, a large percentage of Type 2 diabeticsubjects lose their response to these agents. 63% of Type 2 diabetespatients fail to reach global HbA_(1c) levels of <7% as advised by theAmerican Diabetes Association, and are thus at high risk of developingcomplications. Moreover, almost invariably patients progress through thestages of declining pancreatic function. Insulin treatment is typicallyinstituted after diet, exercise, and oral medications have failed toadequately control blood glucose. The drawbacks of insulin treatment arethe need for drug injection, the potential for hypoglycemia, and weightgain. Consequently there is still an urgent need for novel anti-diabeticagents.

Schaffer et al. used phage display to identify a series of peptidesbinding to two discrete hotspots on the INSR, which showed agonistic orantagonistic activity when covalently linked to form homodimers orheterodimers (Schaffer et al, Proc. Natl. Acad. Sci. USA,100(8):4435-4439, 2003).

A further pharmacological approach for the treatment of Type 2 diabetesis the use of non-peptide small molecules that can activate the INSR, orpotentiate INSR activation by insulin (Moller, Nature 414: 821-827).Such molecules have proved elusive to identify, but two groups havereported examples. L783281 (DMAQ-B1, L7) and its derivative, compound 2,are insulin mimetics identified from a screen for small molecules thatactivate the INSR tyrosine kinase (Zhang et al, Science 284: 974-977,1999; Qureshi et al, J. Biol. Chem. 275(47): 36590-36595, 2000).TLK16998 and TLK19780 are insulin sensitizers identified by theirability to increase autophosphorylation of isolated, naturally expressedhuman INSR (Manchem et al, Diabetes 50: 824-830, 2001; Pender et al, J.Biol. Chem. 277(46): 43565-43571, 2002). Both L783281 and TLK16998potentiate insulin action in insulin-resistant cells by acting on theintracellular portion of the INSR β-subunit, enhancing β-subunitautophosphorylation and subsequent downstream signaling (Li et al,Diabetes 50: 2323-2328, 2001). Compound 2 and TLK16998 have been shownto reduce blood glucose levels in mouse models of diabetes when givencontinuously at high doses (Strowski et al, Endocrinology145(11):5259-5268, 2004; Manchem et al, Diabetes 50: 824-830, 2001).However, none of these compounds appears to have entered clinicaltesting. Agents that target the INSR tyrosine kinase domain are expectedto have side effects due to non-specific activation homologous tyrosinekinase domains in other molecules. The intracellular portion of the INSRβ-subunit is not a suitable target for larger molecules, such asantibodies, which are unable to diffuse into the cell.

Polyclonal autoantibodies from the sera patients with insulin-resistantdiabetes have been identified and used as probes to study insulinaction. These autoantibodies inhibited insulin binding to INSR andbivalent (but not monovalent) forms produced insulin-like biologicaleffects when exposed to tissues in vitro (Kahn et al, Proc. Natl. Acad.Sci. USA 75(9): 4209-4213, 1978; Heffetz and Zick, J. Biol. Chem.261(2): 889-894, 1986).

Jacobs and Cuatrecasas described two rabbit polyclonal antibodies andreported that these antibodies, as well as a number of polyclonalantibodies produced by other investigators, were able to mediate variousinsulin-like effects (Jacobs and Cuatrecasas, CIBA Found. Symp. 90:82-90, 1982).

Kull et al described three mouse monoclonal antibodies, αIR-1, αIR-2 andαIR-3 and a polyclonal, A410, and their use to investigate theimmunochemical cross-reactivity of, and identify the subunits of, theinsulin and somatomedin-C (IGF-1) receptors (Kull et al, J. Biol. Chem.258(10): 6561-6566, 1983). Herrera et al also made antibodies (rabbitpolyclonal anti-INSR peptide antibodies P4 and P5) to study therelationship between the human INSR and IGF-1 receptors (Herrera et al,J. Biol. Chem. 261(6): 2489-2491, 1986).

A positive modulator antibody that increases the on-rate or decreasesthe off-rate of insulin (insulin analog or INSR agonist) for the INSRcould result in an increased residency time of receptor bound insulin(insulin analog or INSR agonist), a change in the rate of INSRinternalization and/or a change in the degree of phosphorylation ofsignaling proteins activated or deactivated by the INSR. These changescould significantly alter both the metabolic and mitogenic activity ofinsulin (insulin analog or INSR agonist) and the level and frequency ofdosing of exogenous insulin (insulin analog or INSR agonist).

A negative modulating antibody that increases the on-rate or decreasesthe off-rate of insulin (insulin analog or INSR agonist) for thereceptor could result in an decreased residency time of receptor boundinsulin (insulin analog or INSR agonist), a change in the rate of INSRinternalization and/or a change in the degree of phosphorylation ofsignaling proteins activated or deactivated by the INSR. These changescould significantly alter both the metabolic and mitogenic activity ofinsulin (insulin analog or INSR agonist) and the level and frequency ofdosing of exogenous insulin (insulin analog or INSR agonist).

It is contemplated that diabetic patients receiving a positivemodulating antibody of the invention would have improvement in bloodglucose levels, glucose tolerance test, and other measures of insulinsensitivity compared to patients not receiving treatment. For example,administration of a positive modulator antibody of the invention isexpected to reduce elevated blood glucose levels toward normal glucoselevels, which are between approximately 70 mg/dL to 125 mg/dL forfasting blood glucose levels according to the American DiabetesAssociation. In one embodiment, administration of an antibody of theinvention reduces blood glucose levels by approximately 15%, 20%, 25%,30%, 35%, 40% or greater compared to a patient not receiving antibodytreatment.

According to the criteria of the World Health Organization and theAmerican Diabetes Association, normal glucose tolerance is defined asglucose levels of below 140 mg per dL measured two hours after ingesting75 g of oral glucose Impaired glucose tolerance is defined as two-hourglucose levels of 140 to 199 mg per dL (7.8 to 11.0 mmol) afteringesting 75-g oral glucose. A patient is said to have impaired glucosetolerance when the glucose level is elevated (compared to a normalhealthy patient) after 2 hours, but less elevated than would qualify fora diagnosis type 2 diabetes mellitus. A patient with impaired glucosetolerance may still have a fasting glucose that is either normal or onlymildly elevated. In one embodiment, administration of an antibody of theinvention reduces two-hour glucose levels (after the 75-g oral glucosedose) by approximately 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or greatercompared to a patient not receiving antibody treatment.

The ADA also recommends a hemoglobin A1c target level of less than 7% inadults. For children, the ADA recommends higher target levels of A1c. Inchildren younger than 6 years old, the recommended level is from 7.5% to8.5%. In children 6 to 12 years old, the recommended level is less than8%. the recommended level for teens 13 to 19 years old, is less than7.5%. A1c is a measure of how well blood sugar levels have remainedwithin a target range over the previous 2 to 3 months. (AmericanDiabetes Association, Diabetes Care, 28(1): 186-212, 2005.) It iscontemplated that administration of an antibody of the invention totreat diabetes reduces A1c levels towards that observed in anon-diabetic individual. In one embodiment, administration of anantibody of the invention reduces A1c levels in a patient by an absoluteHbA1c percentage measurement of at least 0.5%, 0.7%, 1.0% or 1.5%.

Beta cells in the pancreatic islets of Langerhans make and releaseinsulin, a hormone that controls the level of glucose in the blood.There is a baseline level of insulin maintained by the pancreas, but itcan respond quickly to spikes in blood glucose by releasing storedinsulin while simultaneously producing more. The response time is fairlyrapid. In Type 1 diabetes, progressive and extensive loss of beta cellsresults in decreased levels of secreted insulin, eventually leading tohyperglycemia (abnormally high level of glucose in the blood). In Type 2diabetes, beta cells initially compensate for insulin resistance in asubject by increasing insulin output, but, over time, the cells becomeunable to produce enough insulin to maintain normal glucose levels. Itis thought that both resistance of target tissues to the action ofinsulin and decreased insulin secretion, in part due to beta cellfailure, occur. Administration of antibodies or polypeptides describedherein which improve glucose uptake and other diabetic symptoms are alsouseful to improve beta cell function in a subject in need thereof. Suchimprovement includes, but is not limited to, preservation of beta cellviability or reduction of beta cell turnover, increased beta cellproliferation, or enhanced insulin secretion. Additional methods for andresults of improvement of beta cell function are disclosed in co-ownedinternational application no. WO 2010/028273.

In certain embodiments, treatment with a positive modulating antibody orpartial agonist antibody results in an improvement of one, two, three ormore symptoms of diabetes or insulin resistance selected from the groupconsisting of elevated plasma triglycerides, elevated plasmaunesterified cholesterol, elevated plasma total cholesterol elevatedplasma insulin (indicative of insulin resistance), elevated HOMA-IR,high non-HDL/HDL cholesterol ratio (or high total cholesterol/HDLcholesterol ratio), improved beta cell function, and elevated plasmaleptin levels (indicative of leptin resistance). Where elevated levelsare indicative of diabetes, insulin resistance or increased risk forcardiovascular complications, an “improvement” manifests as a reducedlevel, and vice versa. “Improvement” as used herein refers to anormalization of a level toward the level seen in healthy subjects.

Although the “normal” levels determined upon testing vary on alaboratory-by-laboratory basis, and each laboratory has its own normalrange, in general, normal triglyceride levels are less than 150 mg/dl indiabetes (borderline high 150-199 mg/dL); normal cholesterol levels areless than 200 mg/dL, a normal or target non-HDL/HDL cholesterol ratio isapproximately <3.25 (based on <130 mg/dL non-HDL target and >41 ng/dLtarget HDL), a normal or target range for fasting insulin isapproximately 5-20 microU/m, and a normal or target range for leptin(usually also associated with body mass index (BMI) or hyperinsulinemia)is between 3-25 ng/ml, e.g., 3 ng/mL appears to be required for normalmetabolic function and 20-25 ng/mL appears to be associated withdisease. In some embodiments, treatment normalizes any one or more ofthe above symptoms by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50% or more.

Polycystic ovary syndrome (PCOS) is the most common gynecologicalendocrine disorder and is present in approximately 5-10% of women ofchildbearing age. Clinical presentations include menstrual disorders,obesity, infertility and hirsutism. Insulin resistance in PCOS resultsfrom a post-insulin binding defect in signaling. INSR and insulinreceptor substrate (IRS)-1 serine hyperphosphorylation by anunidentified kinase(s) contributes to this defect. Mitogenic signalingwas observed to be enhanced in skeletal muscle from women with PCOS(Corbould et al, Diabetes 55: 751-59, 2006). Agonists and/or positivemodulators of insulin binding to INSR may therefore be useful fortreating and/or reducing the likelihood of the onset of disorders andsymptoms related to PCOS. Agonists and/or positive modulators of insulinbinding to INSR that do not increase the ratio of mitogenic to metabolicsignaling may be particularly useful for treating PCOS.

Non-alcoholic steatohepatitis (NASH) is part of a spectrum of pathology(known as NAFLD) ranging from simple steatosis (fatty infiltration) toNASH, through to cirrhosis and hepatocellular carcinoma (Farrell andLarter, Hepatol. 43, S99-112, 2006). Insulin resistance is associatedwith fat accumulation in the liver and this organ is now recognized as amajor target of injury in patients with insulin resistance. It isestimated that about 20% of all adults have NAFLD, and 2-3% of adultshave NASH. Up to one third of patients with NASH will develop cirrhosisover longer follow up. Liver disease is a significant complication ofType 2 diabetes.

Individuals with obesity and dyslipidemia exhibit poorer insulinsensitivity than that found in the average population. Obesity is achronic disease that is highly prevalent and is associated not only witha social stigma, but also with decreased life span and numerous medicalproblems including adverse psychological development, dermatologicaldisorders such as infections, varicose veins, exercise intolerance,diabetes mellitus, insulin resistance, hypertension,hypercholesterolemia, and coronary heart disease (Rissanen et al.,British Medical Journal, 301: 835-837, 1990). Obesity is highlycorrelated with insulin resistance and diabetes in experimental animalsand humans. Indeed, obesity and insulin resistance, the latter of whichis generally accompanied by hyperinsulinemia or hyperglycemia, or both,are hallmarks of Type 2 diabetes. In addition, Type 2 diabetes isassociated with a two- to four-fold risk of coronary artery disease.Despite decades of research on these serious health problems, theetiology of obesity and insulin resistance is unknown. It is disclosedherein that positive modulator antibodies and partial agonist antibodiescan reduce or slow the weight gain, i.e., normalize weight gain,observed in diabetic animals. It is contemplated that the antibodieshave the same effect on weight gain in obese patients. It has also beendemonstrated that administration of positive modulator antibodies canslow or reduce weight loss, i.e., normalize weight loss, in diabeticanimals whose beta cell population is depleted, which often results insignificant weight loss and wasting.

In some embodiments it is contemplated that administration of positivemodulator antibodies or partial agonist antibodies described herein canreduce or slow weight gain in a subject by at least 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45% or 50% compared to an untreated subject.

In an alternate embodiment, it is contemplated that administration ofpositive modulator antibodies or partial agonist antibodies describedherein can reduce or slow weight loss in an individual, such as adiabetic patient or an individual having at least partial beta celldepletion, by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50%compared to an untreated subject.

In some embodiments, it is contemplated that administration of positivemodular antibodies or partial agonist antibodies described herein canpromote or induce weight loss relative to untreated subjects, e.g. by atleast 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% compared to anuntreated subject.

Protease inhibitors used for the treatment of HIV patients areassociated with development of a group of metabolic disorders, includinginsulin resistance (Graham, JAIDS 25: S4-S11, 2000). HIV proteaseinhibitor-induced insulin resistance may lead to hyperglycemia that canprogress to diabetes and ultimately life threatening ketoacidosis. (Carret al, Lancet 351:1881-1883, 1998). For some patients, these metabolicside-effects greatly limit the use of these life sustaining drugs.Murata et al (J. Biol. Chem. 275(27): 20251-54, 2000) reported that atleast three of the commercialized HIV protease inhibitor drugs alsoinhibit the glucose transporter from localizing to the cell membrane ofin 3T3 L1 adipocytes, with the subsequent inhibition of glucose uptakeby these cells. This inhibition of cellular glucose transport into cellsby these HIV protease inhibitors is consistent with the elevation ofglucose and lipids observed in the clinic for some patients beingtreated with these protease inhibitor drugs. Thus agonists and/orpositive modulators of insulin binding to INSR may be useful fortreating the metabolic side-effects of HIV protease inhibitors.

Insulin resistance is also one of the pathological features in patientswith hepatitis C virus (HCV) infection and plays a crucial role in thedevelopment of various complications and events associated with HCVinfection (Kawaguchi and Sata, World J. Gastroenterol. 16: 1943-52,2010). Thus agonists and/or positive modulators of insulin binding toINSR may be useful for treating complications and events associated withHCV infection.

INSR signaling may also play a role in other diseases. For example, ithas been speculated that INSR/IGF-1R signaling may play a role inamyloid-beta metabolism (Freude et al, Curr. Alzheimer Res. 6(3):213-23, 2009). Activation of IR has been postulated to be an essentialelement of photoreceptor neuroprotection (Rajala et al, J. Biol. Chem.283(28):19781-92, 2008). Insulin signaling has also been suggested topromote bone formation (Rosen and Motyl, Cell 142: 198-200). Treatmentwith insulin sensitizers has been reported to improve pulmonary functionin patients with both chronic obstructive pulmonary disease and diabetesmellitus (Kim et al, Int. J. Tuberc. Lung Dis. 14(3): 362-67, 2010).

A few patients with homozygous mutations in the INSR gene have beendescribed, which causes Donohue syndrome or Leprechaunism. Thisautosomal recessive disorder results in a totally non-functional insulinreceptor. These patients have low set, often protuberant, ears, flarednostrils, thickened lips, and severe growth retardation. In most cases,the outlook for these patients is extremely poor with death occurringwithin the first year of life. Other mutations of the INSR gene causethe less severe Rabson-Mendenhall syndrome, in which patients havecharacteristically abnormal teeth, hypertrophic gingiva (gums) andenlargement of the pineal gland. Both diseases present with fluctuationsof the glucose level: after a meal the glucose is initially very high,and then falls rapidly to abnormally low levels (Longo et al, Hum Mol.Genet. 11(12): 1465-75, 2002).

Therapeutic Indications for INSR Antagonists/Negative Modulators

The INSR has also been implicated in cancer. Several epidemiologicalstudies have shown that insulin resistance states, characterized byhyperinsulinemia, are associated with an increased risk for a number ofmalignancies, including carcinomas of the breast, prostate, colon andkidney. INSR, particularly the INSR-A form, is overexpressed in severalhuman malignancies. INSR forms hybrid receptors with IGF-IR, which isalso commonly overexpressed in cancer. Hybrid receptors containingINSR-A hemidimers have broad binding specificity as they bind IGF-I andalso IGF-II and insulin. By binding to hybrid receptors, insulin maystimulate specific IGF-IR signaling pathways. Antagonists and/ornegative modulators of insulin binding to INSR and/or to hybridINSR/IGF-1R receptors may therefore be useful as novel anti-cancertherapies (Belfiore Current Pharm. Design 13 (7): 671-686, 2007). INSRhas been reported to be essential for virus-induced tumorigenesis ofKaposi's sarcoma (Rose et al, Onogene 26: 1995-2005, 2007).

Hyperinsulinemia is a condition defined by abnormally high levels ofinsulin in the blood. Causes of hyperinsulinemia include insulinoma andinsulin resistance, which may be caused by congenital hyperinsulinemiaor other conditions, such as a lack of activity, obesity, polycysticovary syndrome or insulin overdose. An insulinoma is a tumor of thepancreas that produces excessive amounts of insulin. High insulin levelscause hypoglycemia, or low blood glucose (sugar). Hyperinsulinemia isthe most common cause of neonatal hypoglycemia following the first fewhours of life. Treatment of such a condition may often be necessary toprevent onset of seizures and neurologic sequelae.

Insulin overdose may be caused, for example by: administration of toomuch insulin; by administration of the right amount of insulin but thewrong type, such as of short acting insulin instead of long-actinginsulin; by administration of insulin followed by a failure to eat; orby intentional insulin over-administration.

In general, hypoglycemia may be mild and lead to symptoms such asanxiety and hunger, but patients are also at risk for severehypoglycemia, which can cause seizures, coma, and even death. Typicalsymptoms associated with hypoglycemia that patients complain aboutinclude tiredness, weakness, tremulous and hunger. Many patients have toeat frequently to prevent symptoms from the low blood sugar. Somepatients may develop psychiatric symptoms because of the low bloodsugar.

Currently, patients with insulinomas or other severe forms ofhyperinsulinemia are treated by surgery such as partial pancreatectomyor by administration of drugs such as diazoxide or somatostatin which insome cases reduces insulin production. In some cases glucose must beinfused continuously. Although peptide INSR antagonists have beendescribed (Schaffer et al, BBRC 376: 380-383, 2008), there is noexisting treatment which reduces the effects of circulating insulin.Antagonists and/or negative modulators of insulin binding to INSR may beuseful for stabilizing patients with insulinomas before surgery or aspart of the therapeutic armamentarium Antagonists and/or negativemodulators are also useful to treat Kaposi's sarcoma.

Additionally, a significant number of patients (25,000-100,000) in theUS who undergo dialysis present with hypoglycemia due to renal failure(chronic kidney disease, chronic renal disease, chronic kidney failure,chronic renal failure, established chronic kidney disease) and maybenefit from treatment with an antagonist or negative modulator of INSRdescribed herein.

Antagonists and/or negative modulators of insulin binding to INSR may beuseful for treating and/or reducing the likelihood of the onset ofdisorders and symptoms related to hyperinsulinemia in a subject, such asreducing anxiety, abnormal hunger, abnormal fatigue, overeating,psychiatric symptoms associated with low blood sugar, and/orhypoglycemia (including hypoglycemia-related seizure, coma, and death).Antagonists and/or negative modulators of insulin binding to INSR maytherefore be used to treat various types of persistent hyperinsulinemiaconditions, such as nesidioblastosis (KATP-H1 Diffuse Disease, KATP-H1Focal Disease, or “PHHI”), GDH-H1 (Hyperinsulinism/HyperammonaemiaSyndrome (HI/HA), leucine-sensitive hypoglycemia, or diazoxide-sensitivehypoglycemia), islet cell dysregulation syndrome, idiopathichypoglycemia of infancy, Persistent Hyperinsulinemic Hypoglycemia ofInfancy (PHHI), Congenital Hyperinsulinism, insulinoma, insulinoverdose, hypoglycemia due to renal failure (acute or chronic), andchronic kidney disease, e.g., type III, IV or V.

Diagnostic Indications for INSR Agonists/Positive Modulators

Antibodies specific for insulin receptor have been used as tools todiagnose diabetes. U.S. Pat. No. 7,732,154 describes polyclonalantibodies to insulin receptor subunit A (IR-A) as a diagnostic fordiabetes, and reports that elevated levels of free IR-A were detected insera of diabetes and cancer patients. The INSR antibodies disclosedherein are useful to measure insulin receptor, e.g. soluble insulinreceptor-A, or insulin levels in a sample from a patient to determine ifthe levels of INSR or insulin are indicative of diabetes or insulinresistance in the patient. A subject with altered levels of insulin orinsulin receptor compared to normal acceptable levels of these factorsin an otherwise healthy individual may have or be at risk of diabetes orinsulin resistance. The INSR antibodies disclosed herein are also usefulto measure insulin receptor, e.g. soluble insulin receptor A, or insulinlevels in a sample from a patient to determine if the levels of INSR orinsulin are indicative of cancer in the patient. A subject with alteredlevels of insulin or insulin receptor compared to normal acceptablelevels of these factors in an otherwise healthy individual may have orbe at risk of cancer.

In one embodiment, the invention provides a method of diagnosing insulinresistance or insulin sensitivity using any of the antibodies asdescribed herein. In one embodiment, the method comprises measuringlevels of insulin or insulin receptor, e.g. soluble insulin-receptor-A,in a sample from a subject using an antibody described herein, whereinan altered level of insulin or insulin receptor indicates the subjecthas or is at risk for diabetes, insulin resistance, insulin sensitivityor cancer, and optionally, administering a therapeutic to said subjectwho has or is at risk of diabetes, insulin resistance, insulinsensitivity or cancer. In certain embodiments, the sample is abiological sample. In some embodiments, the biological sample isselected from the group consisting of blood, serum, plasma, urine,papillary secretions, cerebrospinal fluid and tumor biopsy. Methods ofmeasuring insulin receptor in a sample include, but are not limited to,immunoassays, competitive inhibition assays, immunoprecipitation assays,and other assays as described herein.

Assays Useful to Measure the Effects of Modulator Administration

Effects of administration of positive or negative modulator antibodiesto subjects are measured in vivo and in vitro. In one embodiment, it iscontemplated that antibodies that positively modulate insulin/insulinreceptor activity decrease in vivo levels of HbA1c, cholesterol, LDL,triglycerides, or non-esterified fatty acids, and HDL in a subject.These factors are measured using techniques common to those of skill inthe art.

Subjects receiving a positive modulator antibody also may show reducedweight or reduced weight gain, a decreased frequency and/or number ofhypoglycemic or hyperglycemic events, and improved: HDL/LDL ratio,insulin secretion, glycemic control (as measured by glucose tolerancetest GTT)), insulin sensitivity as measured by insulin tolerance test(ITT)), beta-cell function (as measured by, e.g., cell mass, insulinsecretion, C-peptide levels), beta-cell dormancy, dyslipidemia.

Improved insulin resistance is measured by normalized gene expression ofany of the following in liver, adipose tissue and/or muscle: Pck1(PEPCK), G6pc (G6Pase), Srebf1 (SREBP-1), Gck (GK), Ppargc1a (PGC-1),Abca1 (ABC-1), Acaca (acetyl-CoA carboxylase), IL1b (IL-1beta), IL6(IL-6), Tnf (TNF-alpha), Ccl2 (MCP-1), S1c2a4 (GLUT4), Il-1rn (IL-1ra),CD68, SAA1, SAA2, FAS (fatty acid synthase), Emr1 (F4/80), Irs1, Irs2.The above are measured by well-known techniques in the art.

In vitro assays are also useful to measure the effects of administrationof a modulator of insulin/insulin receptor activity. Positive modulatorantibodies are expected to result in increased translocation of GLUT4 tothe cell surface. Methods for measuring the translocation of GLUT4 froman intracellular location to the plasma membrane are provided forexample in U.S. Pat. No. 6,632,924, US 2007/0141635, US 2003/0104490 andLiu et al, Biochem. J. 418(2), 413-20 (2009). Effects of positivemodulators may also be assessed by analyzing enhanced glucose uptake byliver, adipose and/or muscle cells, enhanced depletion of glucose fromliver, adipose and/or muscle cell culture medium, and measuring theratio of metabolic to mitogenic INSR signaling increased or unchanged,pAKT activation, and pIRS-1 activation. The relative Hill slope ofinsulin-INSR interaction is also measurable. Some dose response curves,however, are steeper or shallower than the standard curve. The steepnessis quantified by the Hill slope, also called a slope factor. A doseresponse curve with a standard slope has a Hill slope of 1.0. A steepercurve has a higher slope factor and a shallower curve has a lower slopefactor. Exemplary assays to analyze these factors are described in theExamples.

Use of INSR Antibodies as Drug Delivery Agents

An antibody to INSR, 83-14, has been humanized for the purpose ofcreating a “molecular Trojan horse” to deliver protein and non-viralgene therapies across the blood-brain-barrier. 83-14 binding drivesrapid internalization of the INSR. Hence, further antibodies with thisproperty, or improved properties, may be useful for drug delivery to thebrain and central nervous system (Boado et al, Biotech and BioEng.96(2): 381-391; WO04/050016).

Kits

As an additional aspect, the invention includes kits which comprise oneor more compounds or compositions packaged in a manner which facilitatestheir use to practice methods of the invention. In one embodiment, sucha kit includes a compound or composition described herein (e.g., acomposition comprising a insulin receptor or insulin/insulin receptorcomplex-specific antibody alone or in combination with a second agent),packaged in a container such as a sealed bottle or vessel, with a labelaffixed to the container or included in the package that describes useof the compound or composition in practicing the method. Preferably, thecompound or composition is packaged in a unit dosage form. The kit mayfurther include a device suitable for administering the compositionaccording to a specific route of administration or for practicing ascreening assay. Preferably, the kit contains a label that describes useof the antibody composition.

Additional aspects and details of the invention will be apparent fromthe following examples, which are intended to be illustrative ratherthan limiting.

EXAMPLES Example 1 Isolation of Anti-INSR Antibodies from Antibody PhageDisplay Libraries

(1) Phage Panning and Rescue

A. Naïve Antibody Phage Display Libraries

Human insulin receptor (hINSR) (R&D Systems, MN) was biotinylated withSulfo-NHS-LC-Biotin (Pierce, Rockford, Ill.) using the manufacturer'sprotocol and 16-fold molar excess of biotin reagent. The biotinylationof hINSR was confirmed by surface plasmon resonance (SPR).

For the first round of phage panning, 1.6×10¹¹ cfu of phage particlesfrom an scFv phage display library (BioInvent, Lund, Sweden) wereblocked for 1 h at room temperature (RT) in 1 ml of 5% milk/PBS(Teknova, Hollister, Calif.) with gentle rotation. Blocked phage weretwice deselected for 30 minutes against streptavidin-coated magneticDynabeads® M-280 (Invitrogen Dynal AS, Oslo, Norway). To form thebiotin-hINSR-hINS complex, 100 pmoles of biotinylated hINSR waspreincubated with excess (2,100 pmoles) human insulin (hINS) (Sigma,Mo.) dissolved in 5% milk/PBS, for 1 h at RT with gentle rotation. Forthe second round of panning, 50 pmoles of biotin-hINSR was used with1050 pmoles hINS. For the final round of panning, 25 pmoles ofbiotin-hINSR was incubated with 525 pmoles hINS.

The biotin-hINSR/hINS solution was incubated with blockedstreptavidin-coated magnetic Dynabeads® M-280 (Invitrogen Dynal AS,Oslo, Norway) for 30 minutes with gentle rotation in order to immobilizethe biotin-hINSR-hINS complex. The deselected phage were incubated withthe biotin-hINSR-hINS streptavidin beads for 2 h at RT. In order tosaturate the hINSR with hINS, additional hINS (2,100 pmoles) was addedto the solution. The beads were washed. For the first round of panning,beads were quickly washed (i.e. beads were pulled out of solution usinga magnet and resuspended in 1 ml wash buffer) three times with PBS-0.1%TWEEN, followed by three times with PBS. For the second round ofpanning, beads were quickly washed five times with PBS-0.1% TWEENfollowed by a one 5 minute wash (in 1 ml wash buffer at room temperaturewith gentle rotation) with PBS-0.1% TWEEN and then five times with PBSfollowed by one 5 minute wash with PBS. For the third round of panning,beads were quickly washed four times with PBS-0.1% TWEEN, followed bytwo washes for five minutes with PBS-0.1% TWEEN and then four quickwashes with PBS, followed by two 5 minute washes with PBS.

The hINSR-hINS-bound phage were eluted with 100 mM triethylamine (TEA)(30 min incubation at RT) which was then neutralized with 1M Tris-HCl(pH 7.4). The eluted phage were used to infect TG1 bacterial cells(Stratagene, Calif.) when they reached an OD₆₀₀ of ˜0.5. Followinginfection for 30 min at 37° C. without shaking, and for 30 min at 37° C.with shaking at 90 rpm, cells were pelleted and resuspended in 2YT mediasupplemented with 100 ug/ml ampicillin and 2% glucose. The resuspendedcells were plated on 2YT agar plates with 100 ug/ml carbenicillin and 2%glucose and incubated overnight at 30° C.

Phage was then rescued with helper phage VCSM13 (New England Biolabs,MA) at a multiplicity of infection (MOI) ˜10. Following helper phageinfection at an OD₆₀₀ of 0.6 at 37° C. for 30 min without rotation and30 min incubation at 37° C. at 150 rpm, cell pellets were resuspended in2YT media supplemented with 100 ug/ml ampicillin and 50 ug/ml kanamycinand allowed to grow overnight at 30° C. Phage in the supernatant wererecovered after rigorous centrifugation and used for the next round ofpanning. In order to monitor the enrichment resulting from the phageselections, the amount of input and output phage was titered for thethree rounds of panning.

Gene III Excision and Generation of Bacterial Periplasmic Extracts

Before screening the phage panning output scFv clones for binding to thehINSR-hINS complex, the gene III gene was first excised from thephagemid vectors to enable production of secreted scFv. In order to dothis, a plasmid midi prep (Qiagen, Valencia, Calif.) of the thirdpanning round output pool of clones was digested with the restrictionenzyme EagI (New England Biolabs, MA). The digestion product without thegene III was then allowed to self-ligate with T4 DNA ligase (New EnglandBiolabs, MA) and used to transform chemically-competent TOP10 E. colicells (Invitrogen, Carlsbad, Calif.). Individual transformed colonies in96-well plates were then used to generate bacterial periplasmic extractsaccording to standard methods, with a 1:3 volume ratio of ice-cold PPBsolution (Teknova, Hollister, Calif.) and double distilled water (ddH2O)and two protease inhibitor cocktail tablets (Roche, Ind.). The lysatesupernatants were assayed by ELISA, as described below.

B. Immunized Antibody Phage Display Libraries

An Omniclonal™ phage display library was generated from micehyperimmunized with hINSR-hINS complex according to the methodsdescribed in U.S. Pat. No. 6,057,098. The immunization materialconsisted of approximately equal molar amounts of recombinant humaninsulin (cat #I9278, Sigma-Aldrich, Inc. St. Louis, Mo.) and recombinanthuman INSR (28-956) (cat #1544-IR/CF, R&D Systems, MN). The proteinconcentration of the complex was around 0.24 mg/ml. Single colonies,obtained from the Omniclonal′ library according to the protocol in U.S.Pat. No. 6,057,098, were screened for binding activity in an ELISA assayas described below.

(2) ELISA Screening of Antibody Clones on hINSR/hINS Complex

ELISA Maxisorp® plates (Thermo Fisher Scientific, Rochester, N.Y.) werecoated overnight at 4° C. with 3 ug/ml hINSR in PBS. Plates were thenblocked for 1 h at RT with 400 ul/well 5% milk/PBS. To generate wellscontaining the hINSR-hINS complex, 50 ul/well of hINS (2.1 uM) wasallowed to bind to the hINSR for 30 min at RT. Bacterial periplasmicextracts were also blocked with 5% milk/PBS for 1 h and then added tothe coated ELISA plate (50 ul/well) and allowed to bind to either hINSRor hINSR-hINS complex on the ELISA plate for 2 h at RT. The murine 83-7anti-hINSR mAb was used as a positive ELISA screening control (Soos etal, Biochem. J. 235: 199-208, 1986). Bound scFv fragments were detectedwith murine anti-c-myc mAb (Roche, Ind.) for 1 h at RT followed by goatanti-mouse HRP-conjugated antisera (Thermo Scientific, Rockford, Ill.).Three washes with PBS-0.1% TWEEN-20 (Teknova, Hollister, Calif.) wereperformed following every stage of the ELISA screens. The positivecontrol 83-7 mAb was detected by goat-anti-mouse HRP (Thermo Scientific,Rockford, Ill.) following incubation for 1 h at RT. Color was developedat 450 nm absorbance with 50 ul/well soluble3.3′,5.5′-tetramethylbenzidine (TMB) substrate (EMD chemicals,Calbiochem, N.J.) and stopped with 1M H₂SO₄ (50 ul/well).

Results

ELISA screening of the bacterial periplasmic extracts identifiedmultiple hINSR or hINSR-hINS complex binders that originated from thephage panning selection. Fifty-eight percent (868 out of 1,488) of theclones selected from the naïve library were able to bind the hINSR orhINSR-hINS complex. Forty-three percent (200 out of 465) of the clonesselected from the immunized library were able to bind the hINSR orhINSR-hINS complex. Periplasmic extracts from the selected clones werealso assayed by FACS (see Example 2). Selected clones were reformattedas IgG1 or IgG2 antibodies. The variable heavy (VH) and light (VL)chains of the selected scFv fragments were PCR-amplified, cloned intoplasmid vectors containing antibody constant genes, and transfected into293E EBNA human cells using standard methods.

Example 2 Receptor Occupancy Screen to Determine Antibody Binding toINSR in the Presence or Absence of Human Insulin

This example describes the use of flow cytometric (FACS) based assays tomeasure differential antibody binding to cells in the presence orabsence of human insulin (hINS). Anti-insulin receptor (INSR) antibodiesfrom phage display libraries were screened in the assays to identifymodulators of INS-INSR binding.

IM-9 cells were obtained from the American Type Culture Collection(ATCC) and maintained in RPMI 1640+10% FBS. Prior to use in assays cellswere washed in serum-free RPMI 1640, counted and the concentrationadjusted to 2×10⁶ cells/ml in RPMI 1640+0.5% BSA (Sigma-Aldrich). Thecells were cultured overnight in this media and as such were designatedas “serum-starved.” These cells were washed once and resuspended at2×10⁶ cells/ml in PBS containing 0.5% BSA and 0.01% sodium azide (FACSbuffer).

Cells exposed to insulin were resuspended in FACS buffer supplementedwith 70 nM human insulin (Sigma-Aldrich, St. Louis, Mo.). Both cellpopulations (+hINS) or (−hINS) were incubated at 4° C. for 30 minutes,washed once with FACS buffer and resuspended at 2×10⁶ cells/ml in FACSbuffer. Twenty five microliter aliquots of cells were plated into 96well plates, mixed with 25 ul of antibody or PPE and incubated on icefor 1 h.

The cells were then washed once with FACS buffer and the binding of theantibody was detected by the addition of 25 ul of an appropriatefluorochrome-conjugated secondary antibody. If the initial incubationhad been with PPE containing a myc-tagged antibody, 25 ul of a 1/1000dilution of an anti-c-myc antibody (Roche) was added to the wells andthe cells incubated on ice for 30 mins. The cells were then washed oncewith FACS buffer and the binding of the anti-c-myc revealed by theaddition of a phycoerythrin-conjugated anti-mouse IgG. After a final 15min incubation on ice the cells were washed and the pellets resuspendedin FACS buffer. The cells were analyzed on a FACSCAN™ (Becton-Dickinson,Milipitas, Calif.) and the data analyzed in both FLOWJO™ (Treestar,Ashland, Oreg.) and Microsoft Excel™.

This assay allowed the detection of four types of antibody, examples ofwhich are shown in FIG. 1:

-   1. Antibodies that only bind to IM-9 cells if they have been exposed    to human insulin (bind exclusively to INS/INSR complex)-   2. Antibodies that bind better to IM-9 cells if they have been    exposed to human insulin (bind preferentially to INS/INSR complex)-   3. Antibodies that bind less well to IM-9 cells if they have been    exposed to human insulin (bind preferentially to uncomplexed INSR).

Antibodies were scored as predicted positive modulators if the ratio ofantibody binding to INS/INSR complex: antibody binding to uncomplexedINSR was greater than 1.3. Antibodies were scored as predicted negativemodulators if the ratio of antibody binding to INS/INSR complex:antibody binding to uncomplexed INSR was less than 0.6. Antibodies werescored as predicted non-modulators if the ratio of antibody binding toINS/INSR complex: antibody binding to uncomplexed INSR was greater than0.9 but less than 1.1.

Example 3 Biotinylated Ligand Screen to Determine the Effects ofAnti-INSR Antibodies on Insulin Binding to INSR

This example describes the use of FACS based assays to measuredifferential ligand (human insulin) binding to cells in the presence orabsence of anti-INSR antibodies. Anti-INSR antibodies from phage displaylibraries were screened in the assays to identify modulators of theINS-INSR complex.

IM 9 cells were obtained from the American Type Culture Collection(ATCC) and maintained in RPMI 1640+10% FBS. Prior to use in assays cellswere washed in serum-free RPMI 1640, counted and the concentrationadjusted to 2×10⁶ cells/ml in RPMI 1640+0.5% BSA (Sigma-Aldrich). Thecells were cultured overnight in this media and as such were designatedas “serum-starved.” These cells were washed once and resuspended at2×10⁶ cells/ml in PBS containing 0.5% BSA (binding buffer).

Serum-starved cells were pre-exposed to INSR antibodies at roomtemperature for 15 minutes and then incubated with variousconcentrations of biotinylated human insulin purchased from R&D Systemsfor a further 30 minutes at room temperature. The binding of thebiotinylated insulin was revealed by the addition of a 1/100 dilution ofstreptavidin-phycoerythrin to this mixture for a further 15 minutes atroom temperature. The cells were then washed once with binding bufferand resuspended in equal volumes of PBS containing 0.5% BSA, 0.1% sodiumazide and 2% paraformaldehyde. The cells were analyzed on a FACSCAN™(Becton-Dickinson, Milipitas, Calif.) and the data analyzed in bothFLOWJO™ (Treestar, Ashland, Oreg.) and Microsoft Excel.

FIG. 2 shows the binding of biotinylated insulin to IM9 cells in thepresence or absence of anti-INSR antibodies at different insulinconcentrations. Antibody 83-7 enhanced binding of biotinylated insulin;antibody MA-20 diminished binding of biotinylated insulin; control mouseIgG had no effect on binding of biotinylated insulin.

Example 4 Assay to Determine the Ability of Anti-INSR Antibodies toStimulate pIRS-1 Phosphorylation

The substrate proteins which are phosphorylated by the INSR include aprotein called insulin receptor substrate 1 (IRS-1). IRS-1phosphorylation to form pIRS-1 eventually leads to an increase in thehigh affinity glucose transporter (Glut4) molecules on the outermembrane of insulin-responsive tissues, and therefore to an increase inthe uptake of glucose from blood into these tissues. A pIRS-1 assay wasdeveloped using the Luminex® technology platform (Luminex Corp., Austin,Tex.). Two modes of assay were developed: (a) titration of test antibodyat a fixed concentration of insulin, and (b) titration of insulin at afixed concentration of antibody. Anti-INSR antibodies selected on thebasis of their differential binding to complexed and uncomplexed INSRwere tested in the assays to identify modulators of the INS-INSR complexsignaling.

Cell Treatment and Lysis

IM-9 cells were serum starved for 16-20 hours by counting, centrifuging,washing once with PBS and re-suspending at about 2×10⁶ cells/ml inRPMI+0.5% Sigma Cohn V BSA (10% stock in RPMI, filter sterilized, stored4° C.).

2× concentrated solutions of insulin (Sigma 1-9278 (10 mg/ml) 1.77 mMliquid stock stored at 4′C) dilutions were prepared in RPMI+0.5% BSA. Astandard insulin titration may include 4-fold serial dilutions of forexample: 6.25 nM, 1.56 nM, 0.39 nM, 0.097 nM, 0.024 nM, 0.006 nM, 0.0015nM, 0 nM.

Milliplex MAP Cell Signaling Buffer and Detection Kit (Millipore catalog#48-602) and Phospho-IRS-1 MAP Mates (Millipore catalog #46-627) wereemployed for the detection of pIRS-1 levels, according to themanufacturer's instructions. Briefly, V-bottomed plates containing 50ul/well of 2× treatment media (RPMI containing 0.5% BSA+/− testantibody) were prepared and 1×10⁶ cells serum-starved IM-9 cellsresuspended in 50 ul RPMI+0.5% BSA were added per well. Antibodypretreatment was performed for 15 minutes prior to insulin treatment,either (a) as a bulk antibody/cell mixture at a single antibodyconcentration that was then applied to wells containing serial dilutionsof insulin, or (b) by adding cells directly to wells containing serialdilutions of antibody and spiking in insulin at 0.1 nM. Plates wereplaced in a 37° C. incubator and centrifuged at 1500 rpm at RT for thelast 3 minutes of treatment time (total of 15 minutes). Supernatant wasremoved by inversion and gentle blotting and treated cell pellets werelysed by triturating 3 times using a multi channel pipette with 100 ulLysis Buffer prepared according to Table 4 below (labile components,i.e. protease inhibitors and benzonase, were added just prior to use).Plates were placed on a shaker at RT for 30 minutes and centrifuged at3000 rpm for 10 minutes to clarify the lysate and remove any air bubblesthat may have occurred during trituration. 50 ul of cleared lysate wasremoved and diluted 1:1 in 50 uL Assay Buffer-1 (AB-1) from theDetection Kit, triturated 2-3 times to mix and 50 ul was loaded onto afilter plate membrane on top of the 25 ul/well of diluted beads (seebelow).

TABLE 4 Lysis buffer components 10 20 25 30 40 50 60 100 wells wellswells wells wells wells wells wells Lysis Buffer 1 ml 2 mls 2.5 mls 3mls 4 mls 5 mls 6 mls 10 mls Lysis Buffer 1 2 2.5 3 4 5 6 10 (Milliporecat. # 43-040) SDS 20% stock 0.045 0.09 0.1125 0.135 0.18 0.225 0.270.45 MgCl 50 mM (Invitrogen cat. # 0.02 0.04 0.05 0.06 0.08 0.1 0.12 0.2Y02016) Protease inhibitors 0.02 0.04 0.05 0.06 0.08 0.1 0.12 0.2 (50X)(Millipore cat. # 20-201) Benzonase EMD 0.004 0.008 0.01 0.012 0.0160.02 0.024 0.04 1.01697.0002 @ 250 ug/ml

Filter plate membranes (Millipore Catalog# MABVN1250) were pre-wet with25 ul AB-1/well. Pre-wetting buffer was aspirated from the filter plateusing a Millipore vacuum manifold, being careful not to dry themembranes, and any remaining liquid was blotted from the bottom of thefilter plate. 25 ul of 1× bead suspension was added per well (pIRS-1beads (Millipore catalog #46-627) were pre-prepared by diluting from 20×concentrate into AB-1 buffer and alternately vortexing and sonicatingfor 5 seconds 3 times each).

Filter plate wells were covered with a plate sealer, covered in aluminumfoil to prevent light exposure, and incubated on a plate shaker (setting7-8 on a Labline, Bellco plate shaker or similar model) at either RT for2 hours or alternatively at 4° C. overnight.

Luminex Detection

The filter plates were aspirated and their bottoms blotted. The beadsremained in the well and were washed with 100 ul of AB-1 and placed onshaker for 1-2 minutes. Plates were aspirated, and the wash step wasrepeated.

25 ul per well 1× biotinylated detection antibody, diluted from a 20×stock into AB-1 buffer, was added and plates were incubated on a shakerat RT for 1 hour. Plates were aspirated and their bottoms blotted. 25 ulper well 1× streptavidin phycoerythrin diluted from a 25× stock intoAB-1 buffer, was added and plates were incubated on a shaker at RT for15 minutes. 25 ul of Amplification Buffer (Millipore catalog #48-602)was added to each well, and plates were incubated on a shaker at RT forfurther 15 minutes. The plates were aspirated and the beads wereresuspended in 150 uL AB-1 and read on the Luminex® instrument.

Results

FIG. 3 shows pIRS-1 assay results from titrations of insulin in thepresence of fixed concentrations of representative test antibodies. MFIswere normalized such that the curve fit maximum was adjusted to 100%.Some antibodies (positive modulators) shifted the insulin titrationcurve to the left. Other antibodies (negative modulators) shifted theinsulin titration curve to the right. Varying magnitudes of modulationwere observed. The data in FIG. 3 shows antibodies producing up to a9-fold increase, or up to a 24-fold decrease, in insulin sensitivity.

FIG. 4 shows representative examples of the various functional classesof antibody based on pIRS-1 assay data. In each case results from thetwo modes of assay are shown: (i) titration of insulin at a fixedconcentration of antibody, and (ii) titration of test antibody at afixed concentration of insulin.

FIG. 5 is a table showing insulin EC50 values from the pIRS-1 assay inthe presence or absence of fixed concentrations of various testantibodies. The results are ranked according to EC50 ratio +Ab/−Ab.

Example 5 Measurement of Effects of Anti-INSR Antibodies on INSR-InducedPhosphorylation of AKT and MAPK

The INSR is a tyrosine kinase that undergoes autophosphorylation afterinsulin binding and subsequently catalyzes the phosphorylation ofintracellular proteins such as insulin receptor substrate (IRS) familymembers, Shc, and Gab1. Each of these proteins serves as a docking sitefor the recruitment of downstream signaling molecules resulting in theactivation of various signaling pathways including the PI(3)K/AKT andMAP kinase (MAPK) pathways. These pathways ultimately coordinate toregulate cell growth and differentiation, gene expression, glycogen,protein and lipid synthesis, and glucose metabolism.

The effects of a test antibody on signaling via the INS/INSR complex canbe measured by assessing the ability of the antibody to augmentinsulin-induced serine or tyrosine phosphorylation of specificintracellular proteins, such as AKT and MAPK (ERK1/2), which arespecific to the INSR signaling pathway. The phosphorylation of theseproteins can be measured and quantified by electrochemiluminescence,Western blotting, ELISA, and other techniques known in the art.

In this example assay, CHOK1 cells, engineered to express either thehuman or mouse INSR, were used. These cells were maintained in GrowthMedium containing EX-CELL 302 Serum-Free Medium for CHO Cells(Sigma-Aldrich, St. Louis, Mo.), 2 mM L-glutamine, and 0.4 mg/mLGENETICIN® (Invitrogen, Carlsbad, Calif.). The parental CHOK1 cells wereused as a control and were maintained in Growth Medium withoutGENETICIN®.

On the day before the assay, the cells were washed with PBS, resuspendedat 1×10⁶ cells/mL in Starvation Medium containing RPMI 1640(Invitrogen), 2 mM L-Glutamine, 0.4 mg/mL GENETICIN®, and 0.5% BSA, andincubated for 16-20 hours in a 37° C., 5% CO₂ incubator. The parentalCHOK1 cells were incubated in Starvation Medium without GENETICIN®. Thenext day, cells were resuspended in PBS with 0.5% BSA and 1×10⁵ cellswere added to wells of a 96-well plate. The test antibody was added at0, 1, or 10 ug/ml, approximately 10 minutes prior to the addition ofinsulin. After incubation for 5-60 minutes in a 37° C., 5% CO₂incubator, the treated cells were centrifuged and lysed in a buffercontaining 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA,1% Triton X-100, 10 mM NaF, Phosphatase Inhibitor Cocktails 1 and 2(Sigma-Aldrich), and Complete Mini Protease Inhibitor (Roche DiagnosticsCorporation, Indianapolis, Ind.) for 1 hour with shaking at 4° C. Thelysates were clarified by centrifugation at 485×g for 3 minutes.Electrochemiluminescence using the MesoScale Discovery Multi-spot AssaySystem (Meso Scale Discovery, Gaithersburg, Md.) was used to quantifythe amount of phosphorylated AKT or MAPK present within the lysates.Data were analyzed using GraphPad Prism® (GraphPad Software Inc., LaJolla, Calif.) software to calculate EC50 values from a 4-parameterlogistic equation.

For analysis of agonist activity, the assay was performed as follows. Onthe day before the assay, cells are washed with PBS, resuspended at1×10⁶ cells/mL in Starvation Medium containing RPMI 1640 (Invitrogen), 2mM L-Glutamine, 0.4 mg/mL GENETICIN®, and 0.5% BSA, and incubated for16-20 hours in a 37° C., 5% CO₂ incubator. The parental CHOK1 cells wereincubated in Starvation Medium without GENETICIN®. The next day, cellswere resuspended in PBS with 0.5% BSA and 1×10⁵ cells are added to wellsof a 96-well plate. After incubation with test antibody for 5-60 minutesin a 37° C., 5% CO₂ incubator, the treated cells were centrifuged andlysed in a buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mMEDTA, 1 mM EGTA, 1% Triton X-100, 10 mM NaF, Phosphatase InhibitorCocktails 1 and 2 (Sigma-Aldrich), and Complete Mini Protease Inhibitor(Roche Diagnostics Corporation, Indianapolis, Ind.) for 1 hour withshaking at 4° C. The lysates were clarified by centrifugation at 485×gfor 3 minutes. Electrochemiluminescence using the MesoScale DiscoveryMulti-spot Assay System (Meso Scale Discovery, Gaithersburg, Md.) wasused to quantify the amount of phosphorylated AKT or MAPK is presentwithin the lysates. Data were analyzed using GraphPad Prism® (GraphPadSoftware Inc., La Jolla, Calif.) software to calculate EC50 values froma 4-parameter logistic equation.

FIG. 6 shows pAKT assay results for antibodies representative of: (A)positive modulators (increase insulin-induced signal transduction); (B)positive modulators with agonism (increase insulin-induced signaltransduction and increase insulin-independent signal transduction) (C)non-modulators (no significant effect on insulin-induced signaltransduction); (D) agonists (increase signal transduction independentlyof insulin; may or may not have modulatory activity) (E) negativemodulators (decrease insulin-induced signal transduction). The assayresults also indicate whether the antibodies show functionalcross-reactivity i.e. have effects on both human and mouse INSR-mediatedsignaling.

Example 6 Anti-INSR Antibodies Exhibit a Spectrum of Agonism

The pIRS-1 assay of Example 4 and the pAKT assay of Example 5 were usedto measure the degree of agonism of the selected anti-INSR antibodies.Rather than using titrations of antibodies or insulin, 5 ug/ml anti-INSRantibody was added to the assay in the absence of insulin. The assaymeasured the level of antibody-induced activation of signaling throughINSR in the absence of insulin (agonism).

FIG. 7 shows tabulated results to illustrate that the selectedantibodies exhibit a spectrum of agonism.

Example 7 Change in Cooperativity of Insulin Binding to INSR Effected bya Positive Modulator INSR Antibody

The pAKT assay of Example 5 was performed on one of the positivemodulator antibodies, using various antibody concentrations and adding aserial dilution of insulin. The results are shown in FIG. 8. FIG. 8Ashows there is a dose response of INSR binding in the presence ofdiffering concentrations of antibody and insulin. FIG. 8B shows therelative Hill slope of insulin-INSR interaction in the presence ofvarying concentrations of antibody.

Example 8 Enhancement of Glucose Uptake by a Positive Modulator INSRAntibody

The effects of a positive modulator INSR antibody on glucose uptake in3T3-L1 adipocytes were measured. Upon insulin treatment, INSR isphosphorylated, activating a signal transduction pathway which leads toincreased glucose uptake by glucose transporter 4 (GLUT4) in adipocytes(fat) or myocytes (muscle). Measuring glucose uptake provides a relevantend point assay for insulin sensitivity.

An assay using ³H-2-deoxyglucose as a substrate for GLUT4 was employed(Zen-Bio, Inc., Research Triangle Park, N.C.). Briefly, 3T3-L1preadipocytes were differentiated in 96-well isoplates. Aftermaturation, the cells were washed 2 times with assay buffer and thecells were allowed to rest in Assay buffer for 4 hours. The cells weretreated with anti-INSR antibody, or control antibody (10 ug/ml) andserial concentrations of insulin, or insulin at 0.8 nM for 15 minutes.After 15 minutes glucose uptake was initiated by adding³H-2-deoxyglucose cocktail and the cells were incubated at 37° C., 5%CO₂ for 10 minutes. After 10 minutes, the cells were washed with PBS,lysed, and mixed with scintillation fluid. The CPM of each wellmeasured. Cytochalasin B (10 μM) was used as a negative control.

Results are shown in FIG. 9. FIG. 9 shows the enhancement of insulindependent glucose uptake by a positive modulator antibody. The positivemodulator antibody results in approximately a 2-fold increase in³H-2-deoxyglucose uptake by 3T3-L1 cells in the presence of 10 ug/mltest antibody Ab001 compared to insulin alone.

These results suggest that positive modulator antibodies are useful toinduce glucose uptake in vivo and treat patients exhibiting insulinresistance.

Example 9 Measurement of the Effects of Anti-INSR Antibodies onDepletion of Glucose in Cell Culture Media

Depletion of glucose from cell culture media can be used a surrogatemeasurement of glucose uptake. The effects of anti-INSR antibodies onmedia depletion of glucose is measured as follows.

To measure glucose depletion, a Wako autokit glucose (Cat#439-90901,Autokit C) is used according to the manufactures directions. Briefly,CHOK1 cell lines adapted to adherent with DMEM+10% FBS in 24 or 96 wellplates are plated at an appropriate concentration. The cells are glucoseand serum starved overnight in 0.5% BSA DMEM (no glucose) before use.The starvation media is aspirated and media consisting of the followingis added in the presence and absence of test antibody or isotype controlantibody: group 1, 4 parts DMEM no glucose: 1 part DMEM high glucose(0.9 mg/mL); group 2, 4 parts DMEM no glucose: 1 part DMEM high glucose(0.9 mg/mL)+insulin. At each desired time point, 2 uL samples of mediafrom each well are removed and added to 118 uL of Wako working solution.In some embodiments, sample as are taken at 0, 1, 2-5, 5, 10, and 24hours. Glucose uptake is evaluated with the FLEXSTATION at absorbance505 nm and 600 nM. The amount of glucose is determined as follows:[Average reading for similar samples]/[average reading for standards].Cell count is obtained before and at the end of the experiment tonormalize for cell growth.

Example 10 Measurement of the Effects of Anti-INSR Antibodies on theBalance Between Mitogenic and Metabolic INSR Signaling

INSR signals through two major pathways (1) the PI3 kinase/PDK1/PKBpathway which primarily regulates metabolism, with some influence ongrowth and (2) the Ras/ERK mitogenic pathway which primarily regulatescell growth. The effects of anti-INSR antibodies on the balance betweenmitogenic and metabolic INSR signaling is measured as described in theart. See, e.g., Jensen et al. (Vitam Horm. 80:51-75, 2009), De Meyts andShymko, (Novartis Found. Symp. 227:46-57, 2000); and Rakatzi et al.(Diabetes 52:2227-2238, 2003).

Example 11 Measurement of the In Vivo Effects of Anti-INSR Antibodies

Anti-INSR antibodies found to be cross-reactive with mouse INSR aremeasured in a number of in vivo models. In the DIO model, C57BL/6.1 (B6)male mice (The Jackson Laboratory, Maine) are fed a high fat diet (HFD)for twelve weeks, becoming obese, mildly to moderately hyperglycemic andimpaired for glucose tolerance. This model is used to evaluate theability of INSR antibodies to affect insulin sensitivity in a tightlycontrolled setting. This system also allows a direct comparison of INSRaction and modulation under normal versus diseased conditions. In thisexperiment, DIO or age-matched B6 mice are dosed with INSR antibody 24hours prior to administration of a pre-defined sub-maximal dose ofinsulin in an insulin tolerance test (ITT). Control IgG or maximalinsulin serve as negative and positive controls, respectively.Responsiveness to insulin is assessed by measuring plasma glucose; agreater decrease in glucose over 60 minutes is suggestive of anincreased INSR response. In a separate study, DIO or B6 mice are dosedwith antibody 24 hours prior to a glucose tolerance test (GTT). By thismeasure, lowered fasting glucose and area under the curve (AUC)indicates improved insulin sensitivity.

Two murine models are used to assess the impact of INSR antibodies ontype-2 diabetes progression. ob/ob mice (The Jackson Laboratory, ME) areleptin deficient, becoming obese and only mildly hyperglycemic due tocompensatory hyperinsulemia. In this model, animals receive INSRantibodies beginning at 6 weeks of age or rosiglitazone (PPAR-gammaagonist), an agent previously shown to improve glycemic control in theseanimals. As in the DIO study, glycemic control is assessed by ITT andGTT, every 2 weeks for 6 weeks. In addition, hemoglobin A1c (HbA1c), akey indicator of prolonged elevated plasma glucose, and a lipid panel,is evaluated at the end of the study. In the second model, thestreptozotocin (STZ)/HFD model, pancreatic beta cells in Swiss Albinomice (The Jackson Laboratory, Maine) are ablated via multiple low-doseof streptozotocin, while insulin resistance is induced through HFDfeeding. In this model, animals are severely hyperglycemic due toimpairment of pancreatic insulin output, a situation analogous to latestage T2D (Dakshinamoorty et al, J. Pharm. and Pharmacology 60: 1167-73(2008)). STZ/HFD animals are treated and evaluated in a manner similarto the ob/ob model to measure the effect of INSR antibodies on diseaseprogression.

Example 12 Effects of Partial Agonist Anti-INSR Antibodies on GlycemicControl in DIO Mice

In the diet-induced obesity (DIO) model, C57BL/6 mice can become insulinresistant after approximately 12-14 weeks on a high-fat diet (HFD).Anti-INSR antibodies demonstrated to behave as partial agonists orpositive modulators in vitro were evaluated in this model to determineif these antibodies improved insulin sensitivity and/or glycemic controlin vivo.

To determine whether partial agonist anti-INSR antibodies reduce fastedblood glucose, 20 week-old DIO mice (14 weeks on HFD; n=8/group) werefasted for 5 hours and challenged intravenously with partial agonistantibodies Ab030 and Ab037, or an isotype control (5 mg/kg). Inadditional control studies, DIO mice were treated with insulin (0.5U/kg), or age-matched mice fed a normal diet (ND) were dosed withisotype control (5 mg/kg). Blood glucose was sampled prior to injection(time=0) and 1, 2 and 4 hours post-administration. Compared toage-matched controls, increased blood glucose was observed in DIO mice(HFD-fed/isotype control) at the 1-hour time point, consistent withinsulin resistance in animals fed HFD (FIG. 10A). Administration ofinsulin or either of the partial agonist antibodies resulted in astatistically significant reduction (p<0.05; one-tailed t-test) in bloodglucose (FIG. 10B). Neither antibody induced hypoglycemia at any timepoint (defined as blood glucose <36 mg/dL). These results suggest thatanti-INSR partial agonist antibodies safely and effectively reducefasting blood glucose.

To further evaluate the effect of a partial agonist anti-INSR antibodyon glycemic control, 18-week old DIO mice (12 weeks on HFD; n=8/group)were injected intraperitoneally (IP) with Ab037 (0.1, 1.0 or 9 mg/kg) orisotype control (1.0 mg/kg). As additional controls, age-matched controlmice were dosed with isotype control (1.0 mg/kg) or DIO animals weregiven insulin (0.75 U/kg; IP). A glucose tolerance test (GTT) wasperformed 24 hours after antibody administration (30 min after insulin)by fasting the animals for 16 hours (beginning approximately 8 hoursafter antibody administration), injecting glucose (1.0 U/kg) andfollowing blood glucose over 2 hours. In this experiment, HFD did nothave a significant impact on fasting glucose (FIG. 11B) or post-boluspeak glucose (FIG. 11A). Nevertheless, in DIO mice, partial agonistantibody significantly reduced fasting blood glucose relative to isotypecontrol when dosed at or above 1.0 mg/kg (FIG. 11B) and reduced GTT areaunder the curve (AUC) at 9.0 mg/kg (FIG. 11C).

This outcome demonstrates that an anti-INSR partial agonist antibody canreduce fasting glucose and improve glycemic control in vivo.

Example 13 Effects of Positive Modulator Anti-INSR Antibodies onGlycemic Control in DIO Mice

To determine if a positive modulator anti-INSR antibody improves insulinsensitivity in vivo, 18-week old DIO mice (n=8/group) were given IPinjections of Ab001 (positive modulator)(0.1, 1.0 or 10 mg/kg), partialagonist antibody (Ab037) (10 mg/kg) or isotype control (1.0 mg/kg).Age-matched mice fed ND dosed with isotype control (1.0 mg/kg) served asan additional control (FIG. 12A). Twenty-four hours later, an insulintolerance test (ITT) was carried out by administering insulin (0.5 U/kg)after a 5 hour fast and monitoring blood glucose levels over 2 hours. AHFD did not have a significant impact on fasting glucose (FIG. 12B) orITT AUC (FIG. 12C) relative to regular diet, and neither partial agonistantibody (Ab037) nor positive modulator antibody (Ab001) administrationresulted in a statistically significant lower AUC ITT, relative toisotype control treated DIO animals (FIG. 12C). Partial agonist antibodyAb037 significantly reduced fasting glucose, while positive modulatorantibody Ab001 induced a non-statistically significant, dose-dependenttrend towards reduced fasting glucose.

The following week, a GTT was carried out on the same animals after anadditional dose of antibody (FIG. 13A). In this study, HFD resulted in anon-statistical increase in fasting glucose (FIG. 13B) and GTT AUC (FIG.13C) compared to control fed animals. Compared to isotypecontrol-treated DIO mice, partial agonist antibody and positivemodulator antibody significantly reduced fasting glucose at all dosestested. In addition, both partial agonist antibody and positivemodulator antibody significantly reduced GTT AUC at 10 mg/kg relative toisotype control.

The effect of Ab001 and Ab037 on lipid parameters was investigated bytreating 18-week old DIO mice IP twice weekly (BIW) with antibody (10mg/kg; n=5/group) for twelve weeks. In this experiment, similar efficacyto the two-week study (as described above) was observed with respect tofasting glucose, GTT and ITT. At the end of the study, plasma wascollected to measure lipids using standard ELISA-based techniques.Relative to isotype control, both Ab001 and Ab037 reduced fastingtriglyceride and total cholesterol levels in DIO mice (p<0.05; FIGS. 14Aand 14B), suggesting that these antibodies are able to improve lipiddysregulation associated with insulin resistance.

Two additional positive modulator anti-INSR antibodies were evaluatedfor improvement of glycemic parameters in vivo using 18-week old DIOmice (n=10/group). In this study, positive modulator antibodies Ab083and Ab085 were compared against Ab001 and Ab037 and an isotype controlantibody. A ND fed, age-matched group treated with isotype controlantibody served as an additional control. All antibodies were dosed IPat 10 mg/kg BIW. A day after the third dose of antibody, fasting bloodglucose was measured and a GTT was performed. Glycemic control wassignificantly impaired in isotype control-treated DIO mice relative tosimilarly treated age-matched ND fed animals, as reflected by a GTT timecourse assessment and the corresponding AUC determination (FIGS. 15A and15B). In this experiment, Ab037 and Ab083 improved AUC to levelsindistinguishable from normal (p<0.05 relative to HFD/isotype control),whereas Ab001 did not produce significant improvements. Similarly, withrespect to fasting glucose, a significant difference was observedbetween isotype control treated DIO and age-matched ND-fed mice and bothAb037 and Ab001 exerted statistically significant normalizing effects(p<0.05; FIG. 15C). Ab083 yielded a small, non-statistically significantimprovement in fasting blood glucose, whereas Ab085 did not elicit anychange in this parameter.

Another measure of the effects of anti-INSR antibodies on in vivofunction is by Homeostasis model assessment-insulin resistance(HOMA-IR). HOMA-IR is an empirical mathematical formula based on fastingplasma glucose and fasting plasma insulin levels that was developed as asurrogate measurement of in vivo insulin sensitivity: HOMA-IR=fastingplasma insulin (μIU/mL)×fasting plasma glucose (mmol/L)/22.5, oralternatively using the formula: Insulin (ng/mL)×Glucose (mM), whichincorporates the 22.5 conversion factor. Examples of HOMA-IR aredescribed in Owyang et al., Endocrinology 151:2515-27, 2010 and Matthewset al., Diabetologia. 28:412-9, 1985.

After 4 weeks of dosing, plasma glucose, insulin and lipids wereevaluated. Ab083 and Ab037 reduced plasma glucose at this time point,whereas Ab083 and Ab085 reduced insulin (p<0.05; FIGS. 16A and 16B).These effects translated into improved insulin sensitivity in this modelof insulin resistance for Ab083 and Ab085, as determined by HOMA-IR(p<0.05; FIG. 16C). With respect to lipids, Ab085 significantly improvedonly triglycerides (p<0.05; FIG. 16D), while Ab083 and Ab037significantly reduced unesterified, total and non-HDL cholesterol(p<0.05; FIG. 16E-G). The latter two antibodies also improved thenon-HDL/HDL cholesterol ratio (FIG. 16H). Ab001 significantly reducedboth total and non-HDL cholesterol.

Surprisingly, all four antibodies reduced weight gain in DIO micerelative to isotype control over 3 weeks of treatment, without reducingbody weight to below baseline (FIGS. 17A and 17B). These resultsdemonstrate that the positive modulator antibody Ab083 and agonistantibody Ab037 correct impaired glucose tolerance in DIO mice, thatmodulator antibodies Ab083 and Ab085 improve insulin sensitivity andsuggest that all four antibodies have the capacity to decrease weightgain resulting from HFD.

These results suggest that partial agonist and positive modulatorantibodies specific for the INSR improve glycemic control in diabeticsubjects.

Example 14 Effects of Partial Agonist and Positive Modulator Anti-INSRAntibodies on Glycemic Control and Disease in Db/Db Mice

Mice homozygous for the spontaneous Lepr^(db) allele lack leptinreceptor function and become progressively insulin resistant and obesebeginning at three to four weeks of age. In these mice, insulin levelsrise, until about 8-10 weeks of age, at which time the animals areseverely insulin resistant and hyperinsulinemic. This genetic backgroundnevertheless results in uncontrolled hyperglycemia, leading topancreatic beta cell dysfunction after approximately 10 weeks of age andultimately to beta cell failure. Anti-INSR antibodies demonstrated tobehave as partial agonists or positive modulators in vitro wereevaluated in this model to determine if these antibodies improvedinsulin sensitivity, glycemic control and/or disease progression invivo.

The db/db mouse was utilized to assess the activity of Ab001 and Ab037in a setting of progressive insulin resistance and beta celldysfunction, combined with severe obesity. In this experiment, Ab001 (1mg/kg or 10 mg/kg), Ab037 (10 mg/kg) or isotype control antibody (1mg/kg or 10 mg/kg) were dosed IP, BIW to 5 week-old db/db mice(n=10/group). As an additional control, a group of age-matchedheterozygous littermates, which are generally phenotypically normal,were dosed similarly with 10 mg/kg isotype control antibody. As in theDIO model, weight gain was significantly reduced in animals treated with10 mg/kg of either Ab001 or Ab037 relative to isotype control-treatedmice over the first five weeks of treatment (p<0.05; FIGS. 18A and 18C)Importantly, after 5 weeks of treatment, which corresponds to 10 weeksof age, when db/db mice generally begin losing weight as a result ofpancreatic beta-cell depletion, both antibodies reduce weight loss(p<0.05; FIGS. 18B and 18D). After 10 weeks of treatment, treatment witheither Ab001 or Ab037 at 10 mg/kg significant improvements were observedin fasting blood glucose relative to corresponding isotypecontrol-treated groups (p<0.05; FIG. 19A). In addition, at this timepoint, HbA1c was significantly reduced in the 1 mg/kg Ab001 group, andalso reduced to a lesser degree in the 10 mg/kg Ab001 group (p<0.05;FIG. 19B).

Plasma insulin and lipids were evaluated after 14 weeks of dosing. Atthis time, both Ab001 (10 mg/kg) and Ab037 increased circulating insulinat an age (approximately 20 weeks old) at which these animals would beexpected to have pancreatic beta-cell failure (p<0.05; FIG. 20A),suggesting that both mAbs are capable of restoring insulin output ininsulinopenic animals. In addition, Ab001 (10 mg/kg) significantlyreduced plasma triglycerides, total cholesterol, non-HDL cholesterol,unesterified cholesterol and the non-HDL/HDL cholesterol ratio (p<0.05;FIG. 20B-F). A significant reduction in unesterified cholesterol and atrend towards lowered triglycerides was observed in plasma fromAb037-treated animals (p<0.05 and p=0.08, respectively; FIGS. 20B and20C).

Interestingly, the reduction in weight gain occurred early, while theanimals were insulin resistant, but not expected to have severe betacell depletion, as is the case in the DIO model. However, in thisexperiment, Ab001-induced changes in glycemic control and glycatedhemoglobin occurred only during the late phase, when the animals wouldbe expected to have beta cell dysfunction. Moreover, during this timeperiod, both antibodies reduced pathological weight loss. Not to bebound by theory, this outcome suggests that the anti-INSR antibodyeffects on weight and glycemic control can occur in tandem, but areseparable. These data suggest that Ab001 is capable of normalizingweight, improving glycemic control and partially correcting dyslipidemiaunder conditions of combined insulin resistance and beta cell depletion.

To evaluate the activity of antibodies under severely insulin resistantand insulinopenic conditions, 10-week old db/db mice, which would beexpected to manifest with progressive pancreatic beta cell dysfunction,were treated with Ab001, Ab037, Ab083, Ab085 or isotype controlantibody, at 10 mg/kg IP, BIW for eight weeks. Fasting blood glucose wasmeasured weekly for the duration of the study. In this study, Ab085significantly reduced fasting blood glucose relative to isotype control(p<0.05; FIG. 21). This demonstrates that Ab085 improves disease underinsulin resistant, hypoinsulinemic conditions.

Two additional positive modulator anti-INSR antibodies were evaluatedfor improvement of insulin resistance in 5-week old db/db mice, whichwould be expected to manifest with severe insulin resistance. Mice weretreated with Ab001, Ab037, Ab083, Ab085 or isotype control antibody, at10 mg/kg IP, BIW for four weeks to evaluate the effect of antibodies oninsulin resistance before the onset of beta cell dysfunction. Fastingplasma glucose and fasting plasma insulin were measured at the end ofthe study and HOMA-IR was calculated. In this study, Ab083 and Ab085significantly improved insulin resistance compared to isotype control(p<0.05; FIG. 22), demonstrating that these antibodies improves insulinsensitivity in this model of diabetes.

Example 15 Effects of Partial Agonist and Positive Modulator Anti-INSRAntibodies on Glycemic Control and Disease in MLDS/HFD Mice

In the multi-low dose streptozotocin (MLDS)/HFD model, insulinresistance is achieved by feeding 6-week old ICR mice a HFD (40 kcal %fat) for four weeks, during which time 5 daily doses of streptozotocin(40 mg/kg, during the third week) are administered IP to partiallyablate beta cell function. Anti-INSR antibodies demonstrated to behaveas partial agonists or positive modulators in vitro were evaluated inthis model to determine if these antibodies improved insulinsensitivity, glycemic control and/or disease progression in vivo.

To evaluate the effect of Ab001 and Ab037 on disease in a model ofcombined insulin resistance and beta cell dysfunction, MLDS/HFD mice(n=10/group) were dosed with Ab001, Ab037 or isotype control antibody,at 10 mg/kg IP, BIW for six weeks. One week after the first dose, athree-fold increase in fasting blood glucose was observed in isotypecontrol treated diseased mice, relative to age-matched normal animals,confirming that a diabetic phenotype was achieved. At this time, a GTTwas carried out, revealing significant improvements in glycemic controlfor both Ab001 and Ab037 (p<0.05; FIGS. 23A and 23B). Fasting bloodglucose was also significantly reduced in the group of mice treated withAb037 (p<0.05), whereas no significant change was elicited by Ab001(FIG. 23C). One week later, fed glucose was evaluated Similar to fastingglucose, disease in MLDS/HFD mice was manifested by significantlyelevated fed glucose levels, which was ameliorated by Ab037 (p<0.05;FIG. 24A). Consistent with these improvements in GTT and fed/fastingglucose, Ab037 reduced HbA1c by approximately 1.5% after six weeks ofdosing (p<0.05; FIG. 24B). End of study plasma analysis revealed thatAb037 treatment led to a statistically significant normalization inplasma insulin and a smaller reduction in non-HDL/HDL cholesterol ratio,whereas Ab001 significantly improved plasma leptin levels, with asimilar, but smaller corrective impact on plasma insulin (p<0.05; FIG.25A-C). This model does not present with consistent, disease-relatedweight change as observed in the db/db model, and neither Ab001 norAb037 impacted body weight in this model (FIG. 26), suggesting that thereduced weight gain observed with these antibodies in the other in vivomodels was not a non-specific effect. This data demonstrates Ab037improves multiple manifestations of disease in MLDS/HFD mice, whileAb001 also corrects some parameters of impaired glycemic control in thismodel.

Two additional positive modulator anti-INSR antibodies were evaluatedfor improvement of glycemic parameters in vivo. MLDS/HFD mice(n=10/group) were dosed with Ab001, Ab037, Ab083, Ab085 or isotypecontrol antibody, at 10 mg/kg IP, BIW for six weeks. After 3 weeks oftreatment, a GTT was performed, revealing that Ab037 and Ab083 acompletely normalize glycemic control, relative to isotype control(p<0.05; FIGS. 27A and 27B). Fasting blood glucose was alsosignificantly reduced in mice treated with Ab037 or Ab083 over theduration of the 6-week study (p<0.05), whereas no significant change waselicited by Ab001 or Ab085 (FIG. 28). At the end of the study, plasmalipids were evaluated. Ab083 significantly improved plasmatriglycerides, unesterified cholesterol, total cholesterol, non-HDLcholesterol, non-HDL/HDL cholesterol ratio and free fatty acids (p<0.05;FIG. 29A-F). In addition, Ab001 significantly reduced total, non-HDL andunesterfied cholesterol, as well as non-HDL/HDL cholesterol ratio. Ab037improved non-HDL cholesterol, unesterified cholesterol, non-HDL/HDLcholesterol ratio and free fatty acids. In this experiment, Ab085significantly reduced only free fatty acids. Consistent with theobserved improvements in GTT and fasting glucose, Ab037 and Ab083significantly reduced HbA1c after six weeks of dosing (p<0.05; FIG. 30).In addition, Ab001 and Ab085, which exerted less of an effect on fastingglucose and glucose tolerance, but did improve certain lipid parameters,also reduced HbA1c. As in the previous experiment, none of the mAbsmeaningfully impacted body weight in this model, except Ab085, withreduced weight gain over the first 3 weeks of dosing (FIG. 31). Thisdata demonstrates that all four antibodies tested improve multiplemanifestations of disease in MLDS/HFD mice, without impacting body massin this weight neutral model.

Example 16 Effects of 24 Hour Administration of Partial Agonist andPositive Modulator Anti-INSR Antibodies on INSR Phosphorylation In Vivo

The increase in INSR tyrosine phosphorylation in insulin-sensitivetissues such as liver and muscle by short-term administration ofanti-INSR antibodies confirms that the antibodies are bioavailable andcapable of acting similarly on INSR in vivo as observed in vitro. Inthis experiment, anti-INSR antibodies identified as partial agonists orpositive modulators in vitro were dosed for 24 hours in C56BL/6 malemice and evaluated for their effects on basal and insulin-induced liverand muscle INSR phosphorylation.

To determine if INSR partial agonist and positive modulators increaseINSR phosphorylation in liver and muscle, 10 week-old C56BL/6 male mice(n=3) were given anti-INSR or isotype control antibodies (10 mg/kg) for24 hours, and effects on liver and muscle INSR tyrosine phosphorylationwere determined by ELISA in mice given an insulin bolus (1 U/kg) or PBSfor 10 minutes. Phosphorylated INSR concentrations were normalized tototal insulin receptor concentrations and expressed as a percentage.

Exogenous insulin (1 U/kg) did not significantly increase INSRphosphorylation in control animals (although there was a positive trend)in either liver or muscle (FIGS. 32A, B). However, in liver, significantincreases in insulin-stimulated INSR phosphorylation were observed inAb083- and Ab037-treated mice (p<0.05) as well as a nearly significantincrease in Ab085-treated mice (p=0.07; FIG. 32A). This outcome suggeststhat partial agonist and positive modulator antibodies are capable ofincreasing responsiveness to insulin in vivo. Interestingly, in liver,Ab083 significantly increased INSR phosphorylation even in the basalstate (no exogenous insulin), suggesting that Ab083 is able to sensitizethe response to insulin even in presence of low, fasting levels ofendogenous insulin.

The most pronounced effects from anti-INSR partial agonist and positivemodulator antibodies were seen in the muscle. While all three anti-INSRantibodies positively-modulated insulin signaling in mice receiving aninsulin bolus, Ab083, and to a greater extent, Ab085, also sensitizedmuscle INSR signaling to endogenous, fasting levels of insulin whencompared to control animals (FIG. 32B).

These results suggest that both partial agonist and positive modulatoranti-INSR antibodies improve responsiveness to insulin-mediatedsignaling in liver and muscle in vivo. Relative to the effects of Ab037,antibodies Ab083 and Ab085 sensitize INSR at relatively low insulinconcentrations.

Example 17 Isolation of Anti-INSR Antibodies from Additional AntibodyPhage Display Libraries

Additional naïve antibody libraries were screened for antibodiesspecific for INSR.

(1) Phage Panning and Rescue

Human insulin receptor (hINSR) (R&D Systems, Minneapolis, Minn.) wasbiotinylated as described in Example 1 and used for panning ofadditional naïve antibody phage display libraries.

A. scFv library

scFv Naïve Library: For the first round of phage panning, 4.5×10¹² cfuof phage particles from an scFv lambda phage display library or4.12×10¹² cfu of phage particles from an scFv kappa phage displaylibrary (XOMA LLC, Berkeley, Calif.) were blocked for 1 h at roomtemperature (RT) in 1 ml of 5% milk/PBS (Teknova, Hollister, Calif.)with gentle rotation. This represents two separate pannings, scFv-kappaand scFv-lambda. Blocked phage were deselected twice for 30 minutesagainst streptavidin-coated magnetic Dynabeads® M-280 (Invitrogen DynalAS, Oslo, Norway). To form the biotin-hINSR-hINS complex 103 pmoles ofbiotinylated hINSR was preincubated with excess (2,100 pmoles) humaninsulin (hINS) (Sigma, St Louis, Mo.) dissolved in 5% milk/PBS, for 1 hat RT with gentle rotation. For the second round of panning, 50 pmolesof biotin-hINSR was used with 1050 pmoles hINS. For the final round ofpanning, 25 pmoles of biotin-hINSR was incubated with 525 pmoles hINS.

B. Fab library

Fab Naïve Library: For the first round of phage panning, 1.2×10¹³ cfu ofphage particles or 1.8×10¹³ cfu of phage particles from two differentrescues of an Fab lambda library (XOMA LLC, Berkeley, Calif.), or7.2×10¹² cfu of phage particles or 1.8×10¹³ cfu of phage particles fromtwo different rescues of an Fab kappa library (XOMA LLC, Berkeley,Calif.) were blocked for 1 h at room temperature (RT) in 1 ml of 5%milk/PBS (Teknova, Hollister, Calif.) with gentle rotation. Thisrepresents four separate pannings Blocked phage were twice deselectedfor 30 minutes against streptavidin-coated magnetic Dynabeads® M-280(Invitrogen Dynal AS, Oslo, Norway). To form the biotin-hINSR-hINScomplex, 103 pmoles of biotinylated hINSR was preincubated with excess(2,100 pmoles) human insulin (hINS) (Sigma, St. Louis, Mo.) dissolved in5% milk/PBS, for 1 h at RT with gentle rotation. For the second round ofpanning, 50 pmoles of biotin-hINSR was used with 1050 pmoles hINS. Forthe final round of panning, 25 pmoles of biotin-hINSR was incubated with525 pmoles hINS.

The biotin-hINSR/hINS solution was incubated with blockedstreptavidin-coated magnetic Dynabeads® M-280 (Invitrogen Dynal AS,Oslo, Norway) for 30 minutes with gentle rotation in order to immobilizethe biotin-hINSR-hINS complex. The deselected phage were incubated withthe biotin-hINSR-hINS streptavidin beads for 2 h at RT. In order tosaturate the hINSR with hINS, additional hINS (2,100 pmoles) was addedto the solution. The beads were washed. For the first round of panning,beads were quickly washed (i.e. beads were pulled out of solution usinga magnet and resuspended in 1 ml wash buffer) three times with 0.5%milk-PBS-0.1% TWEEN, followed by three washes with 0.5% milk-PBSfollowed by one quick wash with PBS. For the second round of panning,beads were quickly washed five times with 0.5% milk-PBS-0.1% TWEENfollowed by one 5 minute wash (in 1 ml wash buffer at room temperaturewith gentle rotation) with 0.5% milk-PBS-0.1% TWEEN and then five washeswith 0.5% milk-PBS followed by one 5 minute wash with 0.5% milk-PBS andthen one quick wash with PBS. For the third round of panning, beads werequickly washed four times with 0.5% milk-PBS-0.1% TWEEN, followed by twowashes for five minutes with 0.5% milk-PBS-0.1% TWEEN and then fourquick washes with 0.5% milk-PBS, followed by two 5 minute washes with0.5% milk-PBS and then one quick wash with PBS.

C. Elution and Rescue

The hINSR-hINS-Streptavidin bead-bound phage were eluted with 0.5 ml 100mM triethylamine (TEA) for 30 minutes at RT with gentle rotation. Thebeads were separated from the eluate. The eluate was removed andneutralized with 0.5 ml 1M Tris-HCl (pH 7.4). The beads were neutralizedwith 1 ml 1M Tris-HCl (pH 7.4). The eluted phage from beads or eluate,were used separately to infect TG1 bacterial cells (Stratagene, LaJolla, Calif.) when they reached an OD₆₀₀ of ˜0.5. Following infectionfor 30 min at 37° C. without shaking, and for 30 min at 37° C. withshaking at 90 rpm, cells were pelleted and resuspended in 2YT mediasupplemented with 100 ug/ml carbenicillin and 2% glucose. Theresuspended cells were plated on 2YT agar plates with 100 ug/mlcarbenicillin and 2% glucose and incubated overnight at 30° C.

Phage was then rescued with helper phage M13K07 (New England Biolabs,MA) at a multiplicity of infection (MOI) ˜20. Following helper phageinfection of TG1 cells at an OD₆₀₀ of 0.5 at 37° C. for 30 min withoutshaking and 30 min incubation at 37° C. at 100 rpm, cell pellets wereresuspended in 2YT media supplemented with 100 ug/ml carbenicillin and50 ug/ml kanamycin and allowed to grow overnight at 25° C. and 250 rpm.Phage in the supernatant were recovered after rigorous centrifugationand used for the next round of panning. In order to monitor theenrichment resulting from the phage selections, the amount of input andoutput phage was titered for the three rounds of panning.

(2) FACS Screening of Antibody Clones on Human INSR/hINS or MurineINSR/hINS Complex

Individual colonies were picked and grown in 96-well plates and werethen used to generate bacterial periplasmic extracts according tostandard methods, with a 1:3 volume ratio of ice-cold PPB solution(Teknova, Hollister, Calif.) and ddH2O and protease inhibitor (Roche,Indianapolis, Ind.). The lysate supernatants were assayed by FACS onhINSR/hINS or murine INSR/hINS complex, using the protocol described inExample 2, except that suspension adapted CHO-K1 transfected with eitherhINSR or muINSR were used instead of IM-9 cells, and cells exposed toinsulin were resuspended in FACS buffer supplemented with 150 nM ratherthan 70 nM human insulin. This assay allowed the detection of at least 6types of antibody:

1. Antibodies that only bind to hINSR-CHO cells if they have beenexposed to human insulin (bind exclusively to INS/INSR complex in aspecies specific manner)

2. Antibodies that only bind to muINSR-CHO cells if they have beenexposed to human insulin (bind exclusively to INS/INSR complex in aspecies specific manner)

3. Antibodies that bind to both hINSR-CHO and muINSR-CHO cells if theyhave been exposed to human insulin (bind exclusively to INS/INSR complexin a species cross-reactive manner)

4. Antibodies that only bind to hINSR-CHO cells (bind exclusively toINSR in a species specific manner)

5. Antibodies that only bind to muINSR-CHO cells (bind exclusively toINSR in a species specific manner)

6. Antibodies that bind to both hINSR-CHO and muINSR-CHO cells (bindexclusively to INSR in a species cross-reactive manner)

Antibodies were scored as described in Example 2. Light chain and heavychain sequences of the isolated antibodies were sequenced and are setout in SEQ ID NOs: 87-147 (light chain) and SEQ ID NOs: 223-284 (heavychain).

Results

FACS screening of the bacterial periplasmic extracts identified multipleantibodies that bound human receptor or receptor/ligand complex, hINSRor hINSR-hINS, or murine receptor or receptor/ligand complex, muINSR ormuINSR/hINS. Thirty-three percent (484 out of 1,488) of the clonesselected from these naïve libraries were able to bind the hINSR orhINSR-hINS complex. Twenty-five percent (370 out of 1,488) of the clonesselected from these naïve libraries were able to bind the muINSR ormuINSR-hINS complex. Sixteen percent (234 out of 1,488) of the clonesbound to both hINSR or hINSR-hINS and muINSR or muINSR/hINS complexes byFACS.

Selected clones were reformatted as IgG2 antibodies. The variable heavy(VH) and light (VL) chains of the selected scFv fragments werePCR-amplified, cloned into plasmid vectors containing antibody constantgenes, and transfected into 293E EBNA human cells using standardmethods. Binding of the reformatted antibodies to hINSR or hINSR-hINS ormuINSR or muINSR/hINS were assessed by FACS as described above. Resultsare set out in FIG. 33.

Results show that certain reformatted antibodies bind to both mouse andhuman INSR. FIG. 33 also shows that certain reformatted antibodies binddifferentially to INSR in the presence and absence of insulin and aretherefore predicted to modulate insulin binding to INSR.

Example 18 Panning for Allosteric Agonist Antibodies Against INSR

Selection of agonist antibodies that exhibit greater binding to thecomplex of receptor/ligand than to the free receptor enhances theprobability of identifying antibodies that are noncompetitive with theligand and do not block or diminish binding of the ligand to theorthosteric site of the receptor. An antibody of this type, that bindsto a site on the target receptor distinct from the endogenous bindingsite, is known as an allosteric agonist (Kenakin et al., Journal ofReceptors and Signal Transduction, 27:247-259, 2007; Jahns et al., J AmColl Cardiol. 36:1280-87, 2000; May et al., Ann Rev Toxicol. 47: 1-51,2007).

Methods described above to screen for agonist antibodies are also usefulto screen for allosteric agonists. Preferential binding of the testantibody to the receptor ligand complex is consistent with allostericactivity whereas preferential binding of the test antibody to the freereceptor is consistent with an antibody that competes with insulin forthe orthosteric site. The screen is useful to enrich the pool ofcandidate clones for allosteric agonists by eliminating the some if notall competitive agonists.

Allosteric antibodies are less likely to interfere with the bindingaffinity and efficacy of the ligand and therefore, are less likely tointerfere with the maximum ligand signaling or maximum sensitivity toligand. Allosteric antibodies can exhibit a range of agonism from weakpartial agonists to agonism levels similar to the endogenous ligand. Apartial allosteric agonist will elicit a maximum signaling response thatis of significantly lower in magnitude than the maximum response of theendogenous ligand. In some applications, where sustained sub maximalsignal activation is preferred over maximum signal activation, a partialagonist antibody is preferable to a full agonist antibody. Thedistinguishing characteristics between a partial allosteric agonist anda positive modulator (sensitizer) are evident from a comparison of thedose response curves shown in FIGS. 34 and 17, which show the differentbinding curves for a partial allosteric agonist (FIG. 34) and a positivemodulator (sensitizer) antibody (FIG. 35).

FIG. 34A illustrates an example of the dose response from a partialallosteric agonist in comparison to the dose response to the endogenousligand and FIG. 34B demonstrates activation by ligand in the presence orabsence of the allosteric agonist. FIG. 35A shows the dose response froma positive allosteric modulator antibody in comparison to the doseresponse to the endogenous ligand while FIG. 35B shows a dose responsecurve of an endogenous ligand in the presence and absence of a positivemodulator antibody. FIG. 36 provides the activation parameters for a setof partial allosteric agonists relative to the endogenous ligand. Thenature of signal activation by the partial allosteric agonists isdistinct from that of a positive modulator obtained from the sameprimary screening approach.

A non-competitive partial allosteric agonist antibody may offer atherapeutic advantage over a competitive agonist where it is beneficialto have independent signal activation by both the partial agonist and anendogenous ligand simultaneously. For example, and not to be bound bytheory, a partial allosteric agonist can be used to elevate the basalactivation of a signaling pathway while still allowing response fromtransient fluctuations in endogenous ligand levels. In certaininstances, under conditions where a partial allosteric agonist of thissort is present, the endogenous ligand dose response will exhibit anincrease in the baseline (constitutive or basal) signaling level andwill achieve the same or greater maximal response to the endogenousligand with little or no significant change in the ligand EC50. Forexample, FIG. 34B shows the dose response of an endogenous ligand in thepresence and absence of a partial allosteric agonist and FIG. 37 showsthe maximal activation of insulin in the presence partial allostericagonist antibodies relative to the maximal response to the endogenousligand in the presence of a negative control antibody. FIG. 37demonstrates that the partial allosteric agonist antibodies Ab037 andAb040 have little or no significant impact on the EC50 of the doseresponse and maximum phosphorylation of Akt at Ser473 by insulin whencompared to a negative control antibody within the same assay.

Example 19 Examples of Functional Classes of Anti-INSR Antibodies:Differential Effects on Insulin-Induced Phosphorylation of Akt

The effects of test antibodies on signaling via the insulin/insulinreceptor complex were measured by assessing the ability of theantibodies to sensitize and agonize insulin-induced phosphorylation ofAkt. Assays were performed using the method described in Example 5. Inall data shown, the percent pAtk pSer473 values are relative and do notnecessarily represent absolute cellular pAkt pSer473 levels.

FIGS. 38-40 show pAkt antibody dose response curves in the absence ofinsulin or in the presence of a sub-maximal concentration of insulin forparental CHO-K1 cells, CHO-K1 cells expressing human insulin receptorand CHO-K1 cells expressing mouse insulin receptor. The titrations ofantibodies in the absence of insulin (open symbols) provide anindication of antibody agonism activity. Parallel titration ofantibodies in the presence of a sub-maximal level of insulin (closedsymbols) provides an indication of sensitizing activity relative to theagonism activity. The sensitizing activity can be seen as an increase inthe pAkt levels above that caused by an EC30 concentration of insulin(dashed line) which is greater in magnitude than the agonism activity atthe same antibody concentration. Antibodies Ab077, Ab078 and Ab085 (FIG.38A-C) do not exhibit significant agonism in the absence of insulin.Antibodies Ab001, Ab079 and Ab083 are weak agonists (FIG. 39A-C) andantibody Ab080 shows a moderate level of agonism (FIG. 40). The assayresults also indicate whether the antibodies show functionalcross-reactivity, i.e., have effects on both human and mouseINSR-mediated signaling. Note that antibodies Ab078 and Ab085 only bindthe insulin receptor in the presence of insulin, i.e., they do notdetectably bind unoccupied insulin receptor as assessed by binding toinsulin receptor expressed in CHO-K1 cells in a FACS based assay.

FIG. 41A-C shows insulin induced pAkt activation in the presence offixed concentrations of sensitizing antibodies in comparison to insulinin the presence of IgG2 isotype control antibody anti-KLH (solid lines).pAkt activation levels for antibodies in the absence of insulin at theconcentrations used in the insulin dose response titrations are shown asdashed lines. EC50 values for insulin induced pAkt activation in thepresence of the sensitizing antibodies and fold change in EC50 valuesrelative to isotype control are listed in Table 5.

TABLE 5 EC50 values for insulin induced activation of pAkt in thepresence of sensitizer antibodies. Fold-change in EC50 Antibody relativeto Concentration EC50 isotype Experiment Antibody (ug/ml) (pM) controlHuman INSR Ab001 2 59 12 CHO-K1 cells Ab077 10 81 9 Ab078 20 221 3 Ab07910 100 7 Ab083 2 68 11 Ab085 1 207 3 Ab085 10 81 9 anti-KLH.G2 10 724Mouse INSR Ab001 2 354 7 CHO-K1 cells Ab077 20 301 8 Ab078 20 990 2Ab079 20 300 8 Ab083 10 276 9 Ab085 20 312 8 anti-KLH.G2 10 2414

FIGS. 42A-B show pAkt activation activity of partial allosteric agonistantibodies in the absence of insulin in comparison to insulin alone.Antibodies Ab037, Ab053 and Ab062 all act as agonists of pAkt activityhaving maximal activation plateaus that are significantly less thaninsulin in CHO-K1 cells expressing either human or mouse insulinreceptor. The assay results also indicate whether the antibodies showfunctional cross-reactivity i.e. have effects on both human and mouseinsulin receptor-mediated signaling. Antibody EC50 values and maximumactivation levels are given in Table 6.

TABLE 6 Maximum activation levels and EC50 values for partial allostericagonists Hu Insulin Ab037 Ab053 Ab062 Human INSR Relative 100% 79% 64%52% CHO-K1 maximum activation EC₅₀ (nM) 0.15 0.65 0.42 2.43 Mouse INSRRelative 100% 42% 48% 34% CHO-K1 maximum activation EC₅₀ (nM) 1.70 1.420.69 1.10

FIG. 43 shows insulin dependent pAkt activation in the presence of fixedconcentrations of partial allosteric agonist antibodies in comparison toinsulin alone. Agonist activity of antibodies is seen as an increase inthe baseline of the insulin dose response. The agonist antibodies Ab037and Ab053 have little effect on insulin sensitivity which is reflectedin the lack of significant change in the insulin EC50 and Hillcoefficient in the presence of these antibodies (see Table 7). AntibodyAb062 appears to reduce insulin sensitivity as the EC50 for insulin inthe presence of Ab062 is 6.6-fold higher (see Table 7).

TABLE 7 Insulin activation parameters in the presence and absence ofagonist antibodies Engineered Assay parameter Hu Insulin with insulindetermined 2 ug/ml control Hu Insulin with Hu Insulin with Hu Insulinwith receptor cell from sigmoidal antibody 2 ug/ml Ab037 2 ug/ml Ab053 2ug/ml Ab062 line used in dose-response (95% confidence (95% confidence(95% confidence (95% confidence the assay curve fit interval) interval)interval) interval) Human INSR Relative maximum 100% 109% 99% 106%CHO-K1 activation of pAkt in the (93% to 108%) (106% to 112%) (97% to102%) (100% to 112%) presence of 2 ug/ml antibody EC₅₀ of insulin in the0.58 1.11 0.92 3.91 presence of 2 ug/ml (0.35 to 0.96) (0.84 to 1.5)(0.68 to 1.3) (2.7 to 5.7) antibody (nM) Hill coeff. of insulin in 0.740.79 0.93 0.71 the presence of 2 ug/ml (0.48 to 1.0) (0.63 to 0.95)(0.69 to 1.2) (0.54 to 0.89) antibody

Example 20 Anti-INSR Antibody 83-7 is not a Positive Modulator ofInsulin Binding to hINSR

Anti-INSR antibody 83-7 has been identified previously as specific forhuman insulin receptor, however, the 83-7 antibody has not beendemonstrated to have any modulating abilities on insulin-insulinreceptor binding. In order to assess the ability of the 83-7 antibody tokinetically modulate insulin-insulin receptor interactions,insulin-induced serine phosphorylation of AKT was measured in thepresence of 83-7.

The VH and VL sequences encoding antibody 83-7 (McKern et al., Nature443: 218-221, 2006) were synthesized and the antibody (IgG1, lambdalight chain) was transiently expressed in HEK293 EBNA cells. Theantibody was purified using protein A capture and size exclusionchromatography. The ability of 83-7 to augment insulin-induced serinephosphorylation of AKT was measured using the method described inExample 5. FIG. 44 shows pAKT assay results for 83-7 and Ab001 on CHOK1cells expressing: (A) human INSR, or; (B) mouse INSR. Antibody 83-7 didnot positively modulate insulin-dependent INSR signaling, showing onlyagonist activity on human INSR and did not exhibit agonism on mouseINSR. In contrast, Ab001 positively modulated insulin-dependent INSRsignaling by about 10-fold on both human INSR and mouse INSR.

Example 21 Assay to Measure Modulation of Insulin Binding Affinity forINSR by Anti-INSR Antibodies

To determine the ability of the modulating antibodies to affect thebinding of insulin to the insulin receptor, the affinity of unmodifiedinsulin binding to human INSR expressed on the surface of serum starvedCHOK1 cells (hINSR8-CHOK1) was measured in the presence and absence ofmonoclonal antibodies to INSR. A KinExA assay was developed to measurevery low levels of insulin in cell culture media. This assay allowed thebinding of insulin to cells expressing INSR to be measured bydetermining the level of insulin depletion from the cell culture media.As insulin became bound to the cells, the concentration of insulin inthe cell culture media dropped. By using a titration of cells expressingINSR and measuring the percent free insulin, the affinity of theINS-INSR interaction could be estimated using KinExA software. Thisassay was used to measure the degree of modulation of insulin bindingactivity shown by various anti-INSR antibodies.

hINSR8-CHOK1 cells were serum starved overnight and then prepared forassay by pelleting cells and resuspending at a concentration of 2× thefinal assay concentration for the highest dilutions (between 3.5×10⁷ and2.0×10⁷ cells/mL in assay dilution buffer of PBS (Teknova, HollisterCalif.) with 500 μg/mL BSA and 0.1% sodium azide (Sigma Aldrich, St.Louis, Mo.)). A two-fold serial dilution of cells was prepared creatinga ten-point dilutions series and a no-cells control was also used. Cellsuspensions were aliquoted into polypropylene assay tubes in 2 mL volumeeach. To these cell suspensions 1 mL of 40 ug/mL test antibody (or 100ug/mL for Ab078) was added to each tube, gently mixed and incubated for30-45 minutes on ice. The antibodies used were tested in comparison tothe negative control human IgG2 anti-KLH antibody. 1 mL of 200 pMinsulin was added to each tube to establish a final insulinconcentration of 50 pM (300 pg/mL) (Sigma-Aldrich, St. Louis, Mo.).Samples were incubated overnight at 4° C. for 18 hours then centrifugedto pellet cells and supernatants were removed for testing.

KinExA 3000 analysis was performed using beads coated with ananti-insulin monoclonal antibody. 2 grams of poly(methyl methacrylate)(PMMA) beads (Sāpidyne, Boise, Id.) was suspended in 9 mL of assaybuffer PBS containing 65 ug/mL of clone D6C4 mouse anti-insulinmonoclonal antibody (Fitzgerald Industries, Acton Mass.). Beads wererotated at room temperature for 6 hours then allowed to settle.Supernatant was replaced with PBS with 50 mg/mL BSA Fraction V(Sigma-Aldrich, St. Louis, Mo.) and rotated overnight at 4° C. Detectionsolution used was biotinylated mouse anti-insulin clone D3E7 (FitzgeraldIndustries, Acton Mass.) at 0.15 μg/mL in assay dilution buffer withStreptavidin-PE at 1 ug/mL (Invitrogen, Carlsbad, Calif.). On the KinExA3000 the sample was injected at 0.25 mL/minute for 240 seconds, thenrinsed for 60 seconds in running buffer (PBS with 0.05% sodium azide),then 240 seconds of the detection solution was injected, followed by afinal 90 second wash at 1 mL/minute. The difference in voltage from anearly initial time-point and a time point near the end of the run wasmeasured and used to calculate affinities. The INSR concentration on thecells was estimated at 2.5×10⁵ receptors/cell. Affinity was determinedusing the KinExA software (Sāpidyne, Boise Id.) and EC50's werecalculated by non-linear fit in Prism (GraphPad Software, La JollaCalif.).

A number of anti-INSR antibodies enhanced the affinity of insulin forthe cells. Other antibodies had no effect on insulin affinity for thecells (Table 8). One of the tested antibodies decreased the affinity ofinsulin for the cells by approximately three-fold. FIG. 45 shows freeinsulin percentage plotted against estimated insulin receptorconcentration. The insulin level was fixed at 50 pM and the antibodyconcentration was 10 ug/mL (67 nM) for all clones except Ab078 which wastested at 25 ug/mL (167 nM). Curves shown are the non-linear regressionPrism fit used to calculate EC50.

FIG. 46 shows free insulin percentage plotted against estimated insulinreceptor concentration. The insulin level was fixed at 50 pM and theantibody concentration was 10 ug/mL (67 nM) for all clones. Curves shownare the non-linear regression Prism fit used to calculate EC50.

TABLE 8 Insulin Affinity and IC50 Table K_(D) EC50 Fold Shift Antibody(pM) (pM) in Affinity IgG2-KLH 272 365 1.0 Ab037 271 471 1.0 Ab001 49104 +5.6 Ab053 228 33 +1. Ab062 762 760 22.8 Ab078 41 80 +6.6 Ab079 12.140 +22.5 Ab080 11.2 34 +24.3 Ab083 13.7 39 +19.9 Ab085 34 70 +8.0

Example 22 Assay to Measure Insulin, IGF-1, and IGF-2 MediatedProliferation of MCF-7 Cells in the Presence or Absence of Anti-INSRAntibodies

Insulin, IGF-1, and IGF-2 promote mitogenesis in MCF-7 human mammaryadenocarcinoma cells. Previous studies have shown that insulin analogspromote mitogenic signaling in addition to metabolic signaling followingbinding to INSR. The positive modulator and agonist anti-INSR antibodiesdescribed herein were expected to promote INSR-mediated mitogenicsignaling in parallel to their activation of INSR-mediated metabolicsignaling. The effects of the modulating antibodies on insulin-mediatedmitogenic stimuli were measured using MCF-7 cells expressing thereceptors.

MCF-7 cells were cultured in Dulbecco's Modified Eagles Medium (DMEM)containing glucose at 4.5 g/L supplemented with 10% FBS and 2 mMglutamine (Invitrogen) for normal maintenance. For the proliferationassay, cells were seeded in 96 well white opaque microtiter plates at adensity of 1×10⁴ cells/well (Costar 3917) and allowed to re-attach for24 hrs. After 24 hrs, the cells were washed 2× with pre-warmed PBS andincubated in DMEM containing glucose at 1 g/L and no phenol redsupplemented with 0.1% FBS and 2 mM glutamine (Invitrogen), which willbe referred to as “starvation media,” for another 24 hrs. Insulin(Sigma), IGF-1 (R&D Systems), and IGF-2 (R&D Systems) were prepared as10× stocks in starvation media and serially diluted 5-fold starting from1 uM down to 64 nM (6 dilutions), and added to the cells after the 24 hrstarvation period. For the co-incubation experiments that include theanti-INSR antibodies along with the growth factor, a 50 ug/ml stock ofeach antibody was prepared in starvation media and added to the cellsprior to addition of growth factor to a final concentration of 5 ug/ml.The cells were incubated at 37° C. for 48 hrs and cell proliferation wasmeasured using the CellTiter-Glo Luminescent Cell Viability Assay(Promega). The results are shown in Table 9.

TABLE 9 MCF-7 proliferation results EC₅₀ and 95% confidence intervalvalues Insulin (nM) IGF-1 (nM) IGF-2 (nM) EC₅₀ 95% Cl EC₅₀ 95% Cl EC₅₀95% Cl No antibody 0.80 0.48-1.33 1.73 1.07-2.80 1.30 0.80-2.11 KLH*1.54 1.03-2.29 2.10 1.33-3.30 2.66 1.75-4.06 Ab001* 3.46 2.22-5.40 4.713.28-6.75 2.54 1.87-3.43 Ab037* 1.54 1.00-2.37 2.33 1.51-3.58 2.091.44-3.05 Ab083* 1.08 0.49-2.38 0.91 0.58-1.43 1.47 0.77-2.81 Ab085*0.48 0.28-0.81 2.11 1.38-3.21 1.84 1.30-2.60 *antibody concentration @ 5ug/ml

These results show that, surprisingly, in the presence anti-INSRantibodies, no significant changes in the mitogenic responses to any ofthe aforementioned growth factors were observed within a 95% confidenceinterval. It is possible that these antibodies may cross-react andweakly bind IGF-1 and IGF-2, but the above assay demonstrates that anypossible crossreactive binding does not elicit a functional effect,i.e., does not promote signaling through the receptor. This suggests theantibodies are able to increase the ratio of metabolic to mitogenicINSR-mediated signaling.

Example 23 The Effects of Anti-INSR Antibodies to Reverse InsulinResistant Fatty Acid Uptake in Differentiated 3T3-L1 Adipocytes

TNFα can inhibit insulin dependent fatty acid uptake. Since TNFα isknown to cause insulin resistance by deactivation of insulin signalingpathway intermediates such as IRS-1 (Nguyen et al, J. Biol. Chem.280(42): 35361-71, 2005; Luca and Olefsky, FEBS Let. 582: 97-105, 2008)that are also part of the insulin dependent glucose uptake pathway,reversal of TNFα inhibition of insulin dependent fatty acid uptake byanti-INSR antibodies is indicative of the ability of these antibodies toreverse TNFα mediated inhibition of insulin dependent glucose uptake.

3T3-L1 mouse embryonic fibroblasts can be induced to differentiate intoadipocytes, after which they become highly responsive toinsulin-mediated fatty acid uptake. High fat feeding has beenestablished as a cause of adipose tissue insulin resistance. To examinethis condition in vitro, 3T3-L1 adipocytes have been treated with freefatty acids (FFA) which result in impaired insulin receptor-mediatedsignal transduction and ultimately decreased insulin-stimulated glucoseuptake. One of the downstream effector molecules induced by FFAtreatment that contributes to insulin resistance is TNFα. TNFα has alsobeen shown to inhibit insulin-mediated fatty acid uptake and provides awell defined in vitro system to assess whether anti-INSR antibodies canreverse insulin-resistant fatty acid transport.

3T3-L1 cells were cultured in Dulbecco's Modified Eagles Medium (DMEM)containing glucose at 4.5 g/L supplemented with 10% newborn calf serum(NCS; Invitrogen) and 2 mM glutamine (Invitrogen) for normalmaintenance. To differentiate cells into adipocytes in 96-wellmicrotiter plates, the following protocol was used: (1) at day −5, 2×10³cells per well were seeded in a black/clear bottom 96-well plate (BDFalcon 353948), (2) at day −2, cells reach confluency and are left for 2additional days, (3) at day 0 differentiation mediais added, (4) at day3, media is changed to normal growth media containing 0.425 uM insulin,(5) at day 7 media is changed to normal growth media. To induce insulinresistance, 10× stocks of TNFα (R&D Systems) were prepared in normalgrowth media and added to cells on day 9 of the differentiation process.Working concentrations of TNFα used were between 1-10 ng/ml. Fatty aciduptake was run on cells at day 10. The fatty acid uptake protocol usedwas as follows. Cells were washed in 2× in Hank's Balanced Salt Solution(HBSS; Invitrogen) containing 0.2% fatty acid-free BSA (FAF-BSA; Sigma)and 20 mM HEPES (Invitrogen), and then serum starved in HBSS for 1-2 hrsat 37° C. Anti-INSR antibodies or other relevant controls were addedfrom a 10× stock or HBSS alone and incubate at 37° C. for 30 minutes,and insulin added at dilutions from a 10× stock and incubated at 37° C.for 30 minutes. An equal volume of reconstituted QBT Fatty Acid UptakeAssay (Molecular Devices) loading buffer was then added and incubated at37° C. for up to 3 hours, and the plates read on a fluorescent platereader to measure internalized fluorescent fatty acid analogs.

FIG. 47 shows that TNFα-induces desensitization of insulin-mediatedfatty acid uptake in 3T3-L1 adipocytes in the presence of anti-INSRantibody Ab085. Table 10 shows relative EC50 for the antibodies forfatty acid uptake, demonstrating that Ab085 decreases the EC50 for fattyacid uptake. In the presence of anti-INSR antibody Ab085, theTNFα-induced desensitization of insulin-mediated fatty acid uptake wascompletely reversed back to the untreated control values. Similarresults were observed for Ab083.

TABLE 10 EC₅₀ and 95% confidence interval values Insulin (nM) EC₅₀ 95%CI Insulin only 0.77 0.37-1.60 +TNFα 2.89 1.37-6.08 +TNFα, +anti-KLH3.39 1.42-8.11 +TNFα, +Ab085 0.32 0.14-0.75 TNFα concentration @ 1.25ng/ml Ab085 concentration @ 50 ug/ml

These results demonstrate that the positive-modulator antibody canincrease fatty acid uptake in adipocytes, which suggests the antibody isuseful to treat a disorder or condition that would benefit fromincreasing fatty acid uptake.

Example 24 Characterization of Highly Purified Anti-INSR Antibodies byInsulin Dependent pAkt Activation

A certain amount of assay-to-assay variation was noted in the functionalpIRS-1 and pAKT assays. It was determined that this variation could bereduced when the test antibodies were purified using a further step inaddition to protein-A purification, e.g., size-exclusion chromatography,resulting in antibodies that were approximately >95% pure. Thispurification step reduced or eliminated aggregates and contaminatinggrowth factors thought to interfere with the functional assay.

A number of highly purified anti-INSR antibodies were tested in the pAKTassay described in Example 5, using CHOK1 cells expressing either thehuman INSR or mouse INSR. In addition, certain anti-INSR antibodies weretested for activity on a CHOK1 cell line transfected with cynomolgusmonkey INSR (CHOK1-cynoINSR-4).

The effects of positive modulator anti-INSR antibodies Ab001, Ab037,Ab077, Ab079, AB080, Ab083 were measured in the pAKT assay and resultsare shown in FIG. 48 (human INSR) and FIG. 49 (mouse INSR).

The relative % pAKT of agonist antibodies Ab037, Ab030, Ab053 and Ab062on human INSR and mouse INSR are shown in FIGS. 50 and 51, respectively.

The relative % pAKT of positive modulator antibodies and agonistantibodies were also measured in CHOK1 cells expressing cynomolgusmonkey INSR4. FIG. 52 demonstrates that the anti-INSR antibodies Ab030,Ab037, Ab053, Ab001, Ab079, AB080 and Ab083 are capable of inducing AKTphosphorylation after activation of monkey INSR.

Additionally, the relative % pAKT of negative modulator antibodiesAb061, Ab070 and Ab081 were also measured in CHOK1 cells expressinghuman INSR. The results are shown in Table 11 and FIG. 53.

TABLE 11 20 ug/mL 10 ug/mL 20 ug/mL 20 ug/mL Isotype Ab061 Ab070 Ab081control mAb Insulin EC50 32.43 2.09 4.53 0.12 Fold change in 269 17 38EC50 relative to Isotype control mAb EC50 95% 14.36 to 1.686 to 3.503 to0.09691 to Confidence 73.25 2.598 5.843 0.1496 Intervals

These results demonstrate that negative modulator antibodies increasethe EC50 of insulin, in some cases by several hundred-fold.

Example 25 Assessment of Species Cross Reactivity of Anti-INSRAntibodies

This example describes the use of a FACS based assay to assess thebinding of insulin receptor antibodies to cells of species such asrabbit and cynomolgus monkey that are often used in toxicologicalstudies. Anti-INSR antibodies from phage display libraries were screenedfor both binding to peripheral blood monocytes of human, rabbit andcynomolgus monkeys and for differential binding in the presence orabsence of the ligand (insulin) to the monocytes of the above namedspecies.

Cynomolgus monkey whole blood was obtained from California NationalPrimate Research Center (Davis, Calif.) and rabbit whole blood wasobtained from LifeSource Biomedical, LLC (Moffett Field, Calif.). HumanPBMC were purified using Ficoll Hypaque from buffy coats obtained fromthe American Red Cross. Cynomolgus and Rabbit PBMC were than purifiedusing Ficoll Hypaque gradients (Pharmacia). Purified PBMC were frozenand stored in liquid nitrogen prior to use in the assay. Human,cynomolgus and rabbit PBMC were thawed and washed with FACS Buffer (0.5%BSA and 0.1% NaN3 in PBS). Once the cells were prepared, they were usedin the FACS staining assay at a final concentration of 2×10⁶ cells/ml.

To look at differential binding, cells were incubated in the presence orabsence of the insulin with decreasing concentrations of anti-INSRantibody at 4° C. for 1 hour and washed once with FACS Buffer. Thebinding of anti-INSR antibody was revealed by the addition of goatanti-human IgG Alexa647 (Jackson ImmunoResearch) for 30 minutes at 4° C.After washing twice with FACS buffer, cells were stained with variousmarkers to capture monocytes population. Human and cynomolgus cells werestained with CD45 and CD14. Rabbit cells were stained with CD11b andCD14. Antibodies were than incubated for 20 minutes and washed twicewith FACS Buffer. Cells were than fixed with 2% paraformaldehyde andequal volume of FACS Buffer was added prior to cell analysis. The cellswere analyzed on a FACScan™ (Becton-Dickinson, Franklin Lakes, N.J.) andthe data was analyzed using both FloJo™ (Tristar, Paso Robles, Calif.)and GraphPad Prism 5 (GraphPad Software, La Jolla, Calif.).

The binding seen on human, rabbit or cynomolgus monkey PBMC wasconfirmed by generating CHO cell-lines that expressed the appropriatespecies insulin receptor and repeating the binding assay describedabove. Data shown in FIG. 54 shows that many of the antibodies thatbound to the human insulin receptor also bound to the rabbit and thecynomolgous insulin receptor and that this binding was modulated by thepresence of insulin.

Example 26 Measurement of the Affinity of Anti-INSR Antibodies in thePresence and Absence of Human Insulin

The affinity of various anti-INSR antibodies for recombinant human INSRexpressed on the surface of serum starved CHOK1 cells (hINSR8-CHOK1) wasmeasured in the presence and absence of insulin. A KinExA assay wasdeveloped to measure very low levels of antibody in an incubationbuffer. This assay allowed the binding of antibodies to cells expressingINSR to be measured by determining the level of antibody depletion fromthe incubation buffer. As antibody became bound to the cells, theconcentration of antibody in the buffer solution dropped. By using atitration of cells expressing INSR and measuring the percent freeantibody, the affinity of the antibody to INSR interaction could beestimated using KinExA software. This assay was used to determine therelative affinities of the tested antibody clones in the presence orabsence of insulin and demonstrated insulin-dependent modulation ofantibody binding to the cells.

hINSR8-CHOK1 cells were serum starved overnight and then prepared forassay as described in Example 20. One mL of 4 ug/mL insulin or bufferwas added to each tube of cells to establish a final insulinconcentration of 0 or 175 nM (1 ug/mL). Then 1 mL of 40 ng/mL antibodywas added to each tube to yield a final antibody concentration of 10ng/mL or 66.6 pM. Samples were incubated overnight at 4° C. for 18 hoursthen centrifuged to pellet cells and supernatants were removed fortesting on the KinExA. The KinExA 3000 analysis was performed asdescribed in Example 20 using beads coated with an (Fab′)2 fragment goatanti-human IgG (H+L) (Jackson Immuno Research, West Grove Pa.).Detection solution used was R-PE-(Fab′)2 fragment goat anti-HumanIgG(H+L) (Jackson Immuno Research, West Grove Pa.). For the 83-7 murineantibody the beads were conjugated as above with a rabbit anti-mouseF(ab′)2 antibody (Jackson Immuno Research, West Grove Pa.) and thedetection solution used was an R-PE-(Fab′)2 fragment Goat anti-MouseIgG(H+L) (Jackson Immuno Research). The INSR concentration on the cellswas estimated at 2.5×10⁵ receptors/cell and bivalent antibody binding toINSR was assumed.

The affinities of a number of anti-INSR antibodies in the presence andabsence of insulin are shown in Table 12. The agonist antibodies Ab037,Ab053, and Ab062 have binding that is independent of insulin and showedless than a two-fold affinity shift in the presence or absence ofinsulin. The 83-7 mouse antibody had a modest three-fold affinity shiftin the presence of insulin, where as the positive modulator antibodiesAb001, Ab079, Ab080, and Ab083 all showed positive binding modulation inthe presence of insulin ranging from seventeen-fold for Ab080 to over100-fold for Ab001. The positive modulators Ab077 and Ab078 have aweaker affinity in the absence of insulin than the other clones and, asa result, their “without insulin” affinity was beyond the range of theassay, which is limited in maximum receptor concentration given the useof the cells as a receptor source. Although binding can be seen withthese clones in the absence of insulin, it is substantially weaker thanin the presence of insulin and modulated to a much greater extent than83-7, but the degree of modulation cannot be accurately estimated withthese assay conditions. Ab085 showed little to no evidence of binding inthe absence of insulin and its binding is considered insulin dependent.

TABLE 12 Affinity of Antibodies to hINSR8-CHOK1Cells Fold With WithoutImprovement mAb Insulin Insulin with Insulin Ab001 1.16E−10 1.20E−08 103Ab037 8.00E−11 1.08E−10 1.4 Ab053 9.60E−11 1.48E−10 1.5 Ab062 1.08E−101.24E−10 1.1 Ab077 6.40E−09 Out of Range* Ab078 3.40E−10 Out of Range*Ab079 4.96E−10 9.60E−09 19.4 Ab080 6.80E−10 1.20E−08 17.6 Ab083 3.76E−107.60E−09 20.2 Ab085 2.00E−10 No Binding 83-7 1.60E−10 4.80E−10 3.0

Example 27 Epitope Binning of Anti-INSR Antibodies

A multifactorial approach was taken to epitope binning to determine ifvarious anti-INSR antibodies bind to potentially similar epitopes or ifthey have demonstrated differential binding properties and differentepitope recognition. Competitive binding or “binning” experiments wereperformed as well as analysis of the antibodies' ability to bind todifferent human and murine species of the insulin receptor and theirability to bind in the presence and absence of insulin. All of these arefactors in determining the potential similarity or difference ofantibody binding epitopes. Flow cytometry assays were performed byanalyzing the binding of biotinylated IgG's to serum starvedhINSR8-CHOK1 cells and mINSR-CHOK1 cells in the presence and absence ofinsulin

For the competitive binding assay, hINSR8-CHOK1 cells and mINSR-CHOK1cells were serum starved overnight and then prepared for assay asdescribed in Example 20. In some embodiments, it is useful to calculatethe number of receptors on the cell surface to carry out the competitionbinding assays. For example, hINSR8-CHOK1 receptor expression levelswere determined initially by standard cell staining and flow cytometrytechniques. Briefly this was carried out by staining the cells with asaturating concentration of MA-20 monoclonal Ab (ThermoFisherScientific, Waltham Mass.) and detecting with R-Phycoerythrin conjugatedgoat anti-mouse IgG antibody (Jackson Immuno Research, West Grove Pa.)and then comparing relative fluorescence with BD Quantibrite™ PE Beads(BD Biosciences, Franklin Lakes N.J.) to provide an estimation of numberof Phycoerythrin molecules bound and extrapolate the number of insulinreceptors based on the number of phycoerythrin molecules bound. Thisnumber was then further tested and refined using KinExA as described inRathanaswami et al, (Analytical Biochemistry 373:52-60, 2008). Briefly,KinExA experiments were performed looking at both antibody and insulinbinding where the ligand concentration used was much higher than thatdescribed herein for the determination of affinities which creates amore stoichiometrically limited dose response. This was then analyzed inthe KinExA software (Sapidyne, Boise Id.) using an unknown ligand modeland determination of a ligand multiplier parameter that was used toconfirm binding receptor concentration. In the present assay, forexample, it is estimated that the hINSR8-CHOK1 cells when serum starvedexpress roughly 250,000 tetrameric INSR receptors per cell. For theantibody affinity, this means the stoichiometry of 2 antibodies perreceptor tetramer and for the high affinity insulin binding site, a 1:1tetramer to insulin ratio.

The antibodies to be tested were biotinylated using standard aminechemistry and the activated PEG4-biotin (Thermo-Fisher, Waltham Mass.).Mouse antibodies 83-7 and 83-14 were also tested. These antibodies havebeen reported to bind to amino acids 233-281 of the CR domain and to theFnIII-I domain of INSR, respectively (McKern et al, 2006; Nature 443:218-21). After serum starving the transfected cells overnight, the cellswere stained with a titration of the biotinylated antibodies in thepresence of 1 ug/mL insulin. Antibodies were incubated on cells at 4° C.for approximately 30 minutes. Samples were then washed 2× in FACS bufferand Streptavidin-phycoerythrin (Jackson ImmunoResearch Labs, West Grove,Pa., USA) was used to detect biotinylated antibody. The concentrationsof biotinylated antibodies used in the binning experiment were selectedbased on them having a subsaturating, but still strong, signal to thehuman cell line in the presence of insulin. Once the concentrations ofthe biotinylated antibodies to be used were experimentally determined,the competition assay was performed as below.

The cells were serum starved overnight and then resuspended in cold FACSbuffer with or without 1 ug/mL human insulin. The cells were then mixed1:1 with 60 ug/mL cold or unlabelled competitor antibody and incubatedat 4° C. for approximately 30 minutes establishing a cold Abconcentration of 30 ug/mL. The biotinylated antibodies were then addedin a 1:2 dilution as a 3× concentration and incubated at 4° C. forapproximately 30 minutes. The cells were then washed 2× in FACS bufferand detected with Streptavidin-phycoerythrin and assayed on a FACSanalyzer (Becton Dickinson, San Jose, Calif.).

MFI was compared between the biotinylated antibodies when mixed with anon-binding control antibody or with a competitor antibody. The extentof binding was measured on the human and the murine cell lines and inthe presence or absence of insulin. A matrix approach was used whereeach biotinylated antibody was tested against each cold competitor.Antibodies with the same competition profiles are considered to be inthe same bin. Exemplary bin groupings as presented in Table 13 arederived from the hINSR8-CHOK1 cells in the presence of insulin asvirtually all clones had the strongest binding under those conditions.Clones shown in Table 13 are labeled to reflect other binding propertiessuch as insulin dependence and murine reactivity.

Results of the experiment resulted in approximately seven differentcompetition bins among the anti-INSR antibodies. An antibody with nocompetition is defined as one exhibiting less than 30% competition,partial competition is competition greater than 30% and less than 80%,and complete competition is greater than 80% competition using themethod described above with hINSR8-CHOK1.

The antibodies that map to Bin 1, which are human and murine reactive,exhibited no competition with AB079, AB076, AB083, partial to completecompetition with AB085 and AB086 and complete competition with AB030,AB037, AB053, AB001, AB018, and AB064, AB040.

The antibodies of Bin 2, which are human and murine reactive, exhibitedthe same profile as those antibodies in Bin 1, but demonstrated nocompetition with AB086 and partial competition with AB078.

The antibodies in Bin 3, which bind to both human and murine INSR,showed no competition with Ab062 and Ab086, partial Competition withAb086, Ab064, Ab001, Ab018 and complete competition with Ab079, Ab076,Ab083, Ab080, Ab062, and Ab020, Ab019, Ab088, Ab089.

Bin 4 antibodies, which bind to human receptor only, exhibited nocompetition with Ab062, Ab086, Ab001, Ab018, Ab030, Ab037, Ab064 andcomplete competition with Ab079, Ab076, Ab083, Ab080, Ab062, and Ab020,Ab019, Ab088, Ab089

Bin 5 antibodies exhibit no competition with AB077, AB001, AB018, AB030,AB037, AB079, AB076, AB083, AB019, AB088, AB089, and AB040 and showcomplete competition with AB064, AB062, AB085, and AB078. Theseantibodies react with both human and murine receptor.

Bin 6 antibodies showed complete to partial competition with almost allclones tested. Clone Ab061 had less than 30% competition with Ab019 andclone Ab074 showed no competition with Ab088. These antibodies reactwith both human and murine receptor.

The antibodies grouped in Bin 7 showed no competition with Ab053, Ab064,83-7, Ab019, Ab088, and Ab089, showed partial competition with Ab037,Ab078, Ab083, Ab080, and Ab085, and showed complete competition withAb040, Ab062, Ab030, Ab001, and Ab018. These antibodies react with bothhuman and murine receptor.

Competition Bin 4 which contains the murine 83-7 clone contained all ofthe clones that lacked murine reactivity. The antibody groupingscorrelated with their functional properties. All of the human agonistantibodies grouped into Bin 1. Positive modulator antibodies groupedinto Bins 3 and 5 with the exception of Ab004. The Bin 3 antibodies bindboth INSR-insulin complex and INSR alone, whereas the Bin 5 antibodiesbind INSR-insulin complex but do not bind INSR alone.

TABLE 13 Epitope Bins 1 2 3 4 5 6 7 Ab030 83-14 Ab079 Ab020 Ab078 Ab061Ab004 Ab037 Ab080 Ab019 Ab085 Ab074 Ab053 Ab083 Ab088 Ab077 Ab001 Ab089Ab018 Ab087 Ab086 83-7 Ab062 Human Ab064 Specific Ab040 Agonist Positivemodulator (complex-specific binding) Positive modulator (binds complexedand free INSR) Negative Modulator

Numerous modifications and variations in the invention as set forth inthe above illustrative examples are expected to occur to those skilledin the art. Consequently only such limitations as appear in the appendedclaims should be placed on the invention.

We claim:
 1. An allosteric antibody that binds to insulin receptor, andthat binds a complex comprising insulin and insulin receptor with anequilibrium dissociation constant K_(D) of 10⁻⁵ M or less, and that iscapable of weakening the binding affinity between insulin and insulinreceptor by at least about 3-fold.
 2. The antibody of claim 1, whereinthe binding affinity is any one of K_(A), K_(D), the ratio of on rate tooff rate, or the ratio of off rate to on rate.
 3. The antibody of claim1, wherein the antibody increases the EC50 of insulin signaling activityby about 2-fold to 1000-fold, optionally in a pAKT assay.
 4. Theantibody of claim 1, wherein the antibody comprises a heavy chainvariable region selected from the group consisting of SEQ ID NOs: 279,241, 258, 155, and 228 and a light chain variable region selected fromthe group consisting of SEQ ID NOs: 139, 103, 119, 8, and
 89. 5. Theantibody of claim 1, wherein the antibody comprises (a) the heavy chainvariable region of any of Ab081 (SEQ ID NO: 279), Ab061 (SEQ ID NO:241), Ab070 (SEQ ID NO: 258), Ab020 (SEQ ID NO: 155), Ab052 (SEQ ID NO:228), Ab087 (SEQ ID NO: 169), Ab019 (SEQ ID NO: 160), Ab088 (SEQ ID NO:153), Ab089 (SEQ ID NO: 154), Ab050 (SEQ ID NO: 226), Ab055 (SEQ ID NO:231), Ab057 (SEQ ID NO: 233), Ab063 (SEQ ID NO: 244), Ab065 (SEQ ID NO:246), Ab072 (SEQ ID NO: 260), Ab074 (SEQ ID NO: 262), and the lightchain variable region of any of Ab081 (SEQ ID NO: 139), Ab061 (SEQ IDNO: 103), Ab070 (SEQ ID NO: 119), Ab020 (SEQ ID NO: 8), Ab052 (SEQ IDNO: 89), Ab087 (SEQ ID NO: 18), Ab019 (SEQ ID NO: 1), Ab088 (SEQ ID NO:2), Ab089 (SEQ ID NO: 4), Ab050 (SEQ ID NO: 87), Ab055 (SEQ ID NO: 92),Ab057 (SEQ ID NO: 94), Ab063 (SEQ ID NO: 105), Ab065 (SEQ ID NO: 107),Ab072 (SEQ ID NO: 121), and Ab074 (SEQ ID NO: 123), or (b) all six CDRsof any of Ab081 (SEQ ID NO: 279, 139), Ab061 (SEQ ID NO: 241, 103),Ab070 (SEQ ID NO: 258, 119), Ab020 (SEQ ID NO: 155, 8), Ab052 (SEQ IDNO: 228, 89), Ab087 (SEQ ID NOs: 169, 18), Ab019 (SEQ ID NOs: 160, 1),Ab088 (SEQ ID NOs: 153, 2), Ab089 (SEQ ID NOs: 154, 4), Ab050 (SEQ IDNOs: 226, 87), Ab055 (SEQ ID NOs: 231, 92), Ab057 (SEQ ID NOs: 233, 94),Ab063 (SEQ ID NOs: 244, 105), Ab065 (SEQ ID NOs: 246, 107), Ab072 (SEQID NOs: 260, 121), and Ab074 (SEQ ID NOs: 262, 123).
 6. The antibody ofclaim 1 that exhibits greater than or equal to 70% competition with anyone, two, three or all antibodies selected from the group consisting ofAb061 (SEQ ID NO: 241, 103), Ab074 (SEQ ID NOs: 262, 123), Ab081 (SEQ IDNO: 279, 139), Ab020 (SEQ ID NOs: 155, 8), Ab019 (SEQ ID NOs: 160, 1),Ab087 (SEQ ID NOs: 169, 18), Ab088 (SEQ ID NOs: 2, 153), and Ab089 (SEQID NOs: 154, 4), optionally wherein the antibody binds human receptor orcomplex and not murine receptor or complex.
 7. The antibody of claim 1,further comprising a human IgG1, IgG2, IgG3, or IgG4 heavy chainconstant region.
 8. The antibody of claim 7, further comprising a humanlight chain constant region.
 9. The antibody of claim 1, wherein saidantibody is a monoclonal antibody.
 10. The antibody of claim 1, whereinsaid antibody is a human antibody.
 11. The antibody of claim 1, whereinthe antibody is conjugated to a hydrophobic moiety.
 12. A method ofpreparing a sterile pharmaceutical composition, comprising adding asterile pharmaceutically acceptable diluent to an antibody of claim 1.13. A sterile composition comprising the antibody of claim 1 and asterile pharmaceutically acceptable diluent.
 14. The antibody of claim 1wherein the variable heavy chain is set out in SEQ ID NOs: 279, 258 or241 and the variable light chain is set out in SEQ ID NOs: 139, 119 or103.
 15. The antibody of claim 1 comprising three heavy chain CDRs setout in SEQ ID NOs: 279, 258 or 241 and three light chain CDRs set out inSEQ ID NOs: 139, 119 or
 103. 16. The antibody of claim 1 wherein thevariable heavy chain is set out in SEQ ID NO: 279 and the variable lightchain is set out in SEQ ID NO:
 139. 17. The antibody of claim 1comprising three heavy chain CDRs set out in SEQ ID NO: 279 and threelight chain CDRs set out in SEQ ID NO: 139.